Antimicrobial kinocidin compositions and methods of use

ABSTRACT

The present invention provides novel kinocidin peptides comprising a C-terminal portion of a kinocidin, wherein the C-terminal portion encompasses an α-helical secondary structure and further displays antimicrobial activity. The kinocidin peptides of the invention are derived from and correspond to a C-terminal portion of a kinocidin that includes a γκo core and that can be a CXC, CC, or C class chemokine. Structural, physicochemical and functional properties of this novel class of antimicrobial peptides and amino acid sequences of particular kinocidin peptides are also disclosed. The invention also provides related antimicrobial methods.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of Ser. No. 16/121,425, filed Sep. 4,2018, now U.S. Pat. No. 10,329,336, which is a continuation of Ser. No.15/226,643, filed Aug. 2, 2016, now abandoned, which is a continuationof U.S. application Ser. No. 12/947,793, filed Nov. 16, 2010, now U.S.Pat. No. 9,428,566, which is a divisional of Ser. No. 12/438,923, filedOct. 6, 2009, now abandoned, which is a U.S. national stage ofInternational Application No. PCT/US2007/014499, filed Jun. 20, 2007,which claims the benefit under 35 U.S.C. § 119(e) from U.S. ApplicationNo. 60/815,491, filed Jun. 20, 2006. The contents of the foregoingapplications are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 10, 2018, isnamed 244713_US2_SEQ_ST25.txt and is 126,599 bytes in size.

BACKGROUND OF THE INVENTION

This invention relates to peptides having antibacterial and antifungalproperties. The invention also concerns the preparation of thesepeptides and compositions containing the same which may be used inagriculture and for human or animal therapy.

Nature provides a context in which organisms across the phylogeneticspectrum are confronted by potential microbial pathogens. In turn,natural selection provides a corresponding requirement for rapid andeffective molecular stratagems of host defense against unfavorablemicrobial infection. Antimicrobial peptides represent a key result ofthis co-evolutionary relationship. While higher organisms have evolvedcomplex and adaptive immune systems, virtually all organisms rely uponprimary innate immune mechanisms that are rapidly deployed to ward offmicrobial invasion. Discoveries over the last decade indicate thatantimicrobial peptides elaborated by essentially all organisms playintegral roles in these innate mechanisms of antimicrobial host defense.

Antimicrobial peptides may be generally categorized as those with orwithout disulfide bridges. Those that contain disulfides commonly adoptβ-sheet structures, while those lacking cysteine crosslinkages oftenexhibit α-helical conformation. Antimicrobial peptides from both classeshave a number of conserved features that likely contribute to theirtoxicity to microorganisms, including: 1) small size, typically rangingfrom 12-50 amino acids; 2) cationicity, with net charges ranging from +2to +7 at pH 7; and 3) amphipathic stereogeometry conferring relativelypolarized hydrophilic and hydrophobic facets (Yeaman and Yount,Pharmacol. Rev. 55:27 (2003)). The limited size of these polypeptidesplaces restrictions on the structural repertoire available to meet theserequirements. Despite these limitations, as a group antimicrobialpeptides display a high degree of variability at non-conserved sites,with amino acid substitution rates on the order of those associated withpositive selection (A. L. Hughes, Cell. Mol. Life Sci. 56:94 (1999)).These observations are consistent with the hypothesis thatco-evolutionary selective pressures drive host-pathogen interactions (M.J. Blaser, N. Engl. J. Med. 346:2083 (2002)).

Amino acid sequence motifs have previously been identified withincertain antimicrobial peptide subclasses (e.g., the cysteine array incertain mammalian defensins; White et al., Curr. Opin. Struct. Biol.5:521 (1995)). Yet, comparatively little is known about morecomprehensive relationships uniting all antimicrobial peptides.Conventional sequence analyses performed have yielded limited sequenceconservation, and no universal structural homology has been identifiedamongst antimicrobial peptides. If present, such a consensus motifacross the diverse families of antimicrobial peptides would provideinsights into the mechanism of action of these molecules, yieldinformation on the evolutionary origin of these sequences, and allowprediction of antimicrobial activity in molecules recognized to haveother functions.

The ability of certain bacteria such as M. tuberculosis and S. aureusamong others, to develop resistance to antibiotics represents a majorchallenge in the treatment of infectious disease. Unfortunately,relatively few new antibiotic drugs have reached the market in recentyears. Methods for administering new classes of antibiotics mightprovide a new scientific weapon in the war against bacterial infections.

There are only a handful of antifungal drugs known for the treatment ofmammals. In fact, there were only ten FDA approved antifungal drugsavailable in 2000 for the treatment of systemic fungal infections. Thereare three important classes of fungal drugs for the treatment ofsystemic infections: polyenes, pyrimidines, and azoles. The FDA has alsoapproved certain drugs belonging to other classes for topical treatmentof fungal infections. Certain traditional antifungal drugs may have asignificant toxicity, and certain antifungal drugs available for use intreatment have a limited spectrum of activity. Still further, certainantifungal drugs among the azoles can have interactions withcoadministered drugs, which can result in adverse clinical consequences.As with the antibiotics, certain fungi have developed resistance tospecific antifungal drugs. Patients with compromised immune systems(e.g., AIDS) patients have in some cases had prolonged exposure tofluconazole for both prophylactic and therapeutic purposes. In 2000,increased use of the drug fluconazole correlated with the isolation ofincreasing numbers of resistant infectious fungi among AIDS patients.Methods of using a new class of antifungal drugs could make newtreatments for fungal infections possible.

Invasive mycoses are very serious infections caused by fungi found innature and which become pathogenic in immunocompromised personsImmunosuppression may be the result of various causes: corticotherapy,chemotherapy, transplants, HIV infection. Opportunistic fungalinfections currently account for a high mortality rate in man. They maybe caused by yeasts, mainly of Candida type, or filamentous fungi,chiefly of Aspergillus type. In immunosuppressed patients, failure ofantifungal treatment is frequently observed on account of its toxicity,for example, treatment with Amphotericin B, or the onset of resistantfungi, for example resistance of Candida albicans to nitrogenderivatives. It is, therefore, vital to develop new antifungal medicinalproducts derived from innovative molecules. In this context,antimicrobial peptides offer an attractive alternative.

Antimicrobial peptides are ubiquitous in nature and play an importantrole in the innate immune system of many species. Antimicrobial peptidesare diverse in structure, function, and specificity. A number ofantimicrobial peptides occur naturally as “host-defense” compounds inhumans, other mammals, amphibians, plants and insects, as well as inbacteria themselves. Synthetic antimicrobial peptides have also beendescribed, including highly amphipathic peptides whose amino acidsequences are related to or derived from the sequences of various viralmembrane proteins.

The significant advantage of peptide antimicrobials resides in theglobal mechanism of their anti-microbial action; because peptides havean inherent capacity to bind and penetrate biological membranes, thesecompounds act by physically disrupting cellular membranes, usuallycausing membrane lysis and eventually cell death. Organisms such asbacteria have little ability to combat this physical mechanism andacquire resistance.

Thus, there exists a need for employing multidimensional proteomictechniques to determine structural commonalities amongst peptideselaborated in phylogenetically diverse organisms—microbial to human—andexplore the potential convergence of structural paradigms in thesemolecules. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

The present invention provides novel kinocidin peptides comprising aC-terminal portion of a kinocidin, wherein the C-terminal portionencompasses an α-helical secondary structure and further displaysantimicrobial activity. The kinocidin peptides of the invention arederived from and correspond to a C-terminal portion of a kinocidin,wherein the kinocidin includes a γ_(KC) core and can be a CXC, CX₃C, CC,or C class chemokine. Structural, physicochemical and functionalproperties of this novel class of antimicrobial peptides and amino acidsequences of particular kinocidin peptides are also disclosed. Theinvention also provides related antimicrobial methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows conventional antimicrobial peptide structure classificationand distribution. Relationship amongst structure and predominance issummarized for the commonly recognized antimicrobial peptide classes.Concatenation represents the proportionate distribution of peptidesencompassing a given structural class, as calculated from theAntimicrobial Sequences Database. Numbers of peptides classified in eachgroup are indicated in brackets for each class.

FIG. 2A shows multiple sequence alignment of antimicrobial peptidesexamined. The MSA of the β-sheet peptide study set was generated usingthe Clustal W tool (Version 1.81; Higgins and Sharp, Gene 73:237 (1988);Higgins and Sharp, Comput. Appl. Biosci. 5:151 (1989)), as visualizedwith Jalview (M. Clamp, Jalview—java multiple alignment editor, version1.7b (1998). Public domain (www.ebi.ac.uk/jalview/)). The colorationscheme is formatted to the Clustal degree of conservation. Individualpeptides are designated by the following information series: peptidename, (source genus), and [Swiss Protein accession code]: protegrin 1,(Sus), [3212589] (SEQ ID NO: 43); gomesin, (Acanthoscurria), [20664097](SEQ ID NO: 44); drosomycin, (Drosophila), [2780893] (SEQ ID NO: 45);MGD-1, (Mytilus), [12084380] (SEQ ID NO: 46); tachyplesin I,(Tachypleus), [84665] (SEQ ID NO: 47); mytilin A, (Mytilus), [6225740](SEQ ID NO: 48); sapecin, (Sarcophaga), [20151208] (SEQ ID NO: 49);HNP-3, (Homo), [229858] (SEQ ID NO: 50); Ah-Amp1, (Aesculus), [6730111](SEQ ID NO: 51); AFP-1, (Aspergillus), [1421258] (SEQ ID NO: 52); mBD-8,(Mus), [15826276] (SEQ ID NO: 53); thanatin, (Podisus), [6730068] (SEQID NO: 54); and gaegurin-1, (Rana), [1169813] (SEQ ID NO: 55).

FIG. 2B shows convergence in the sequence patterns ofcysteine-containing antimicrobial peptides. The consensus primarystructural motifs were identified amongst the prototypicaldisulfide-containing antimicrobial peptide study set. Sequence data anddisulfide arrays indicated were derived from the following sources (indescending order): α-defensins (SEQ ID NO: 56) (Yount et al., J. Biol.Chem. 274:26249 (1999)); β-defensins (SEQ ID NO: 57) (Yount et al., J.Biol. Chem. 274:26249 (1999)); insect defensins (SEQ ID NO: 58)(sapecin; Hanzawa et al., FEBS Lett. 269:413 (1990)); insect CS-αβpeptides (SEQ ID NO: 59) (drosomycin; Landon et al., Protein Sci. 6:1878(1997)); plant CS-αβ peptides (SEQ ID NO: 60) (Ah-AMP-1; Fant et al.,Proteins 37:388 (1999)); crustacea CS-αβ peptides (SEQ ID NO: 61)(MGD-1; Yang et al., Biochemistry 39:14436 (2000)); gaegurin (SEQ ID NO:64) (gaegurin-1; Park et al., Biochem. Biophys. Res. Commun. 205:948(1994)); protegrin (SEQ ID NO: 62) (protegrin-1; Fahrner et al., Chem.Biol. 3:543 (1996)); gomesin (SEQ ID NO: 65) (Silva et al., J. Biol.Chem. 275:33464 (2000); Mandard et al., Eur. J. Biochem. 269:1190(2002)); thanatin (SEQ ID NO: 66) (Mandard et al., Eur. J. Biochem.256:404 (1998)); tachyplesin (SEQ ID NO: 67) (tachyplesin I; Nakamura etal., J. Biol. Chem. 263:16709 (1988)); mytilin (SEQ ID NO: 63) (mytilinCharlet et al., J. Biol. Chem. 271:21808 (1996)); AFP-1 (SEQ ID NO: 68)(Campos-Olivas et al., Biochemistry 34:3009 (1995)). The primarysequences corresponding to the γ-core motif are outlined in red (seeFIG. 4). Sequences are shown in their conventional dextromericorientations (N- to C-termini from left to right) unless indicated to beprojected in a levomeric orientation (levo; C- to N-termini from left toright).

FIG. 3A-H shows conservation of 3-dimensional signatures amongstantimicrobial peptides. Three-dimensional structural alignments werecarried out by the combinatorial extension method (Shindyalov and.Bourne, Protein Eng. 11:739 (1998)), visualized using Protein Explorer(Martz, Trends Biochem. Sci. 27:107 (2002)). Comparisons are between(Ah-AMP-1 ([1BK8], Aesculus, horsechestnut tree) and (peptide name, [PDBaccession code], genus, common name; RMSD): protegrin-1 ([1PG1], Sus,domestic pig; RMSD 1.2 Å; panels A and B); drosomycin ([1MYN],Drosophila, fruit fly; RMSD 1.4 Å; panels C and D); HNP-3 ([1DFN]; Homo,human; RMSD 3.2 Å; panels E and F); and magainin-2 ([2MAG]; Xenopus,frog; Gesell et al., J. Biomol. NMR 9:127 (1997); RMSD 2.6 Å; panels Gand H). Respective amino- and carboxy-termini are indicated in panels A,C, E and G. Panels A, C, E, and G use the Clustal degree of 2° structureconservation coloration scheme. Panels B, D, F, and H employ the DRuMSpolarity-2 color scheme, in which hydrophobic residues are colored gray,while hydrophilic residues are colored purple. By convention, cysteineresidues are indicated as hydrophilic, although in these peptides, theyare oxidized (cystine) and colored gray indicating hydrophobicity Amino-(N-) and carboxy- (C-) termini for comparative peptides are denoted asN1 or N2 and C1 or C2, respective of peptides designated 1 or 2.Relative positions of the disulfide bonds are indicated as dotted yellowlines in panels A-H. See Table II for additional references. Proteinswere visualized using Protein Explorer as described by Martz, TrendsBiochem. Sci. 27, 107-109 (2002).

FIG. 3I-L demonstrates the absence of the γ-core signature innon-antimicrobial peptides. Three-dimensional conformity betweenprototypic antimicrobial and non-antimicrobial peptides was determinedas described in FIG. 3 A-H. Representative comparisons are between theantimicrobial peptide Ah-AMP1, and the following non-antimicrobialpeptides (identified as formatted in FIG. 3 A-H): allergen-5 ([2BBG],Ambrosia, ragweed; RMSD 6.5 Å; panel I); metallothionein II ([1AOO],Saccharomyces, yeast; RMSD 5.3 Å; panel J); TGF-α ([3TGF], Homo, human;RMSD 4.7 Å; panel K); and ferredoxin ([2FDN], Clostridium, bacterium;RMSD 7.4 Å; panel L). Each non-antimicrobial comparator peptide (blue)is shown in maximal alignment with Ah-AMP1 (gray) Amino- (N) andcarboxy- (C) termini are indicated as defined in FIG. 3 A-H. See TableII for references.

FIG. 4 shows conservation of the γ-core motif amongstdisulfide-containing antimicrobial peptides. The conserved γ-core motif(red) is indicated with corresponding sequences (GXC or CXG-C motifs aredenoted in red text). Examples are organized into four structural groupsrelative to the γ-core. Group (γ): protegrin-1, [1PG1] (SEQ ID NO: 69);gomesin [1KFP] (SEQ ID NO: 70); tachyplesin-1 [1MA2] (SEQ ID NO: 71);RTD-1 [1HVZ] (SEQ ID NO: 72); thanatin [8TFV] (SEQ ID NO: 73); hepcidin[1M4F]) (SEQ ID NO: 74); Group (γ-α): sapecin [1LV4] (SEQ ID NO: 75);insect defensin A [1ICA] (SEQ ID NO: 76); heliomicin [1I2U] (SEQ ID NO:77); drosomycin [1MYN] (SEQ ID NO: 78); MGD-1 [1FJN] (SEQ ID NO: 79);charybdotoxin [2CRD] (SEQ ID NO: 80); Group (β-γ): HNP-3 [1DFN] (SEQ IDNO: 81); RK-1 [1EWS] (SEQ ID NO: 82); BNBD-12 [1BNB] (SEQ ID NO: 83);HBD-1 [1E4S] (SEQ ID NO: 84); HBD-2 [1E4Q] (SEQ ID NO: 85); mBD-8 [1E4R](SEQ ID NO: 86)); and Group (β-γ-α): Ah-AMP-1 [1BK8] (SEQ ID NO: 87);Rs-AFP-1 [1AYJ] (SEQ ID NO: 88); Ps-Def-1 [1JKZ] (SEQ ID NO: 89);γ-1-H-thionin [1GPT] (SEQ ID NO: 90); γ-1-P-thionin [1GPS] (SEQ ID NO:91); and brazzein [1BRZ] (SEQ ID NO: 92). Protegrin, gomesin,tachyplesin, RTD-1, and thanatin γ-core sequences (Group γ) are depictedin levomeric orientation. Other peptide data are formatted as in FIG. 3.See Table II for additional references.

FIG. 5A-C shows iterations of the 3-dimensional γ-core motif. Amino acidconsensus patterns of the three γ-core sequence isoforms are shown.Coloration represents the most common residue (>50% frequency) at agiven position, as adapted from the RASMOL schema: cysteine (C), yellow;glycine (G), orange; lysine or arginine, royal blue; serine orthreonine, peach; leucine, isoleucine, alanine or valine, dark green;aromatic, aqua; and variable positions (<50% consensus), gray.

FIG. 6A-I shows molecules exemplifying structure-based or activity-basedvalidation of the multidimensional signature model. Representativemolecules retrieved using the enantiomeric sequence patterns wereidentified (Table III) and analyzed for presence or absence of a γ-coremotif as described. Thus, appropriate molecules were identified tochallenge each of the respective model-based predictions.Three-dimensional structures visualized using Protein Explorer areindicated for: brazzein ([1BRZ], Pentadiplandra, J'Oblie berry, panelsA, D, and G; Caldwell et al., Nat. Struct. Biol. 5:427 (1998));charybdotoxin ([2CRD], Leiurus, scorpion, panels B, E, and H; Bontems etal., Biochemistry 31:7756 (1992)), tachyplesin I ([1MA2], Tachypleus,horseshoe crab, panels C, F, and I); and metallothionein II (see FIG.3). As in FIG. 3, comparative panels A-C use the Clustal degree of 2°structure conservation coloration scheme. Panels D-F employ the DRuMSpolarity-2 color scheme, in which hydrophobic residues are colored gray,and hydrophilic residues are colored purple. As in FIG. 4, amino acidscomprising the γ-core motifs are highlighted in red (panels G-I) withinthe 3-dimensional structures of these representative peptides. Otherdata are formatted as in FIG. 3.

FIG. 7A-H shows experimental validation of the predictive accuracy ofthe multidimensional signature model. Standard radial diffusion assayswere conducted using 10 μg of specified peptide: defensin HNP-1 (HNP);brazzein (BRZ); charybdotoxin (CTX); or metallothionein II (MTL).Recombinant brazzein reflecting the published 3-dimensional structure(1BRZ) as determined by nuclear magnetic resonance spectroscopy waskindly provided by Drs. J. L. Markley and F. M. Assadi-Porter, theUniversity of Wisconsin 25. Charybdotoxin, metallothionein II, anddefensin HNP-1 were obtained from commercial sources. Antimicrobialactivity was assessed using a well-established solid-phase diffusionmethod as described by Tang et al., Infect. Immun. 70: 6524-6533 (2002).Assays included well characterized organisms: Staphylococcus aureus(ATCC 27217, Gram-positive coccus); Bacillus subtilis (ATCC 6633,Gram-positive bacillus); Escherichia coli (strain ML-35, Gram-negativebacillus); and Candida albicans (ATCC 36082, fungus). In brief,organisms were cultured to logarithmic phase and inoculated (10⁶ colonyforming units/ml) into buffered molecular-biology grade agarose at theindicated pH. Peptides resuspended in sterile deionized water wereintroduced into wells formed in the underlay, and incubated for 3 h at37° C. Nutrient-containing overlay medium was then applied, and assaysincubated at 37° C. or 30° C. for bacteria or fungi, respectively. After24 h, zones of complete or partial inhibition were measured. All assayswere repeated independently a minimum of two times at pH 5.5 (panelsA-D) or pH 7.5 (panels E-H) to assess the influences of pH on peptideantimicrobial activities versus microorganisms. Histograms express mean(±standard deviation) zones of complete (blue) or incomplete (yellow)inhibition of growth. These data establish the direct antimicrobialactivities of brazzein and charybdotoxin. Metallothionein II lackedantimicrobial activity under any condition assayed. Note differences inscale.

FIG. 8A shows phylogenetic relationship amongst structural signatures inprototypical antimicrobial peptides. Relative evolutionary distances areindicated at branch nodes in this average distance dendrogram (Saito andNei, Mol. Biol. Evol. 4:406 (1987)). Representative peptides for whichstructures have been determined are (descending order): AFP (AFP-1;Aspergillus, fungal); PRG1 (Protegrin-1; Sus, domestic pig); GOME(Gomesin; Acanthoscurria, spider); THAN (Thanatin; Podisus, soldierbug); HNP3 (Human neutrophil peptide-3; Homo, human); MGD1 (MGD-1;Mytilus, mussel); SAPE (Sapecin; Sarcophaga, flesh fly); MBD8 (Murineβ-defensin-8; Mus, mouse); DMYN (Drosomycin; Drosophila, fruit fly);Ah-AMP1 (AMP-1; Aesculus, horsechestnut tree). Color schema are theClustal degree of 2° structure conservation. These data illustrate theconcept that the γ-core is the common structural element in thesepeptides, suggesting it is an archetype motif of the antimicrobialpeptide signature (see FIG. 4).

FIG. 8B shows modular iterations of multidimensional signatures indisulfide-stabilized antimicrobial peptides. Distinct configurationsintegrating the γ-core are found in naturally occurring antimicrobialpeptides from diverse organisms. Specific examples are used toillustrate this theme (modular formulae are as described in the text):[γ], Protegrin-1; [γα₁], MGD-1; [γβ₁], HNP-3; and [γα₁β₁], Ah-AMP-1.Color schema and peptide identification are as indicated in FIG. 3 (A,C, E, G).

FIG. 9 shows conservation of the multidimensional signature indisulfide-containing antimicrobial peptides. This triple alignmentdemonstrates the dramatic 3-dimensional conservation in antimicrobialpeptides from phylogenetically diverse species spanning 2.6 billionyears of evolution: fruit fly (Drosophila; [1MYN]), mussel (Mytilus;[1FJN]) and horsechestnut tree (Aesculus; 1 BK81). The striking degreeof 3-dimensional preservation reflects a unifying structural codeamongst these broad classes of disulfide containing host defenseeffector molecules. Alignment was carried out using the Vector AlignmentSearch Tool (VAST) available through the National Center forBiotechnology Information (NCBI). Secondary structure is indicated bythe CN3D coloration schema: sheet, gold; helix, green; turn/extended,blue.

FIG. 10A-E, depict amino acid sequences of γ-core signature motifsamongst disulfide-containing antimicrobial peptides. Nomenclature andcoloration are as indicated in FIGS. 2 and 3 of the primary manuscript;standard abbreviations are used for peptide names where appropriate.Lavender shading of molecule identities in Groups IID and IIIB indicatespeptides aligned in the levomeric orientation. These sequencescorrespond to the γ-core pattern map as depicted in FIG. 3 of theprimary manuscript. FIG. 10A depicts amino acid sequences of SEQ ID NOS:93-132, in order from top to bottom. FIG. 10B depicts amino acidsequences of SEQ ID NOS: 133-173, in order from top to bottom. FIG. 10Cdepicts amino acid sequences of SEQ ID NOS: 173-210, in order from topto bottom. FIG. 10D depicts amino acid sequences of SEQ ID NOS: 211-238,in order from top to bottom. FIG. 10E depicts amino acid sequences ofSEQ ID NOS: 239-255, in order from top to bottom.

FIG. 11A-B show peptides with predicted antimicrobial activity based onthe multidimensional signature. Candidate peptides were identified byVAST alignment, 3D-RMSD, and manual comparisons; all RMSD scorescompared with Ah-AMP-1 (1BK8; Aesculus); threshold typically >4.5excluded; each sequence is identified by NCBI accession number. FIG. 11Adepicts amino acid sequences of SEQ ID NOS: 256-390, in order from topto bottom. FIG. 11B depicts amino acid sequences of SEQ ID NOS: 291-315,in order from top to bottom.

FIG. 12 shows alignment of C, CC and CXC class human chemokines (SEQ IDNOS: 316-351). The highlighted GX₃C motif [glycine (G), orange; cysteine(C), yellow; proline (P), aqua] corresponds to the γ_(KC) core signature(outlined in red). Conserved cysteine residues beyond the γ_(KC) coreare shaded gray. Gaps were introduced to achieve maximal alignment; *indicates truncated sequence.

FIG. 13 demonstrates conservation of the γ-core domain within kinocidins(γ_(KC) core). Recurring iterations of the γ_(KC) core motif (red) areindicated with corresponding sequences (GX₃C) denoted in red or goldtext. A comparator antimicrobial peptide (Ah-AMP-1) is also shown toillustrate structural similarities between the γ_(KC) motif and thatpresent in antimicrobial peptides (γ_(AP)). Proteins were visualizedusing protein explorer (Martz, E. (2002) Trends Biochem Sci 27, 107-9).Amino acid sequences of SEQ ID NOS: 352-358 are depicted.

FIG. 14 shows solid-phase antimicrobial activity of human kinocidins andIL-8 subdomains IL-8α and IL-8γ. Peptides (0.5 nmol) were introducedinto wells in agarose plates buffered with MES (2.0 mM, pH 5.5) or PIPES(10.0 mM, pH 7.5). Antimicrobial activity was assessed as the zone ofcomplete (blue) or partial (red) inhibition around the well.Abbreviations are: Native IL-8 (IL-8); IL-8α, (α); IL-8γ, (γ);IL-8α+IL-8γ (α+γ); RANTES, (RAN); GRO-α, (GRO); MCP-1, (MCP);lymphotactin, (LYM); platelet factor-4, (PF-4); and HNP-1, (HNP).Histograms are means±SEM (minimum n=2).

FIG. 15 shows solution-phase microbicidal activity of native IL-8 andsubdomains IL-8α and IL-8-γ. One million CFU of the indicatedmicroorganism per milliliter were incubated with peptide (0.00125-20.0nmol/ml) in either MES (2.0 mM, pH 5.5) or PIPES (10.0 mM, pH 7.5) forone hour at 37° C. Surviving CFU were enumerated and are described aschange in the initial log 10 CFU. ∘, S. aureus ATCC 27217; ●, S.typhimurium strain 5990s; □, C. albicans ATCC 36082. Data are means±SD(minimum n=2).

FIG. 16A-B show spectroscopy for IL-8 structural domains. Spectra weredetermined for the IL-8γ and IL-8α peptides (0.1 mM) in sodium phosphate(10.0 mM, pH 5.5) or PIPES (10.0 mM, pH 7.5) buffer. [ . . . ], (IL-8α);[______], (IL-8-γ_(KC)).

FIG. 17A-B show computational modeling of IL-8 structural domains.Three-dimensional models of IL-8α (A) and IL-8γ (B) peptides werecreated using homology and energy-based methods. Model peptidealpha-carbon backbones were visualized using PyMOL (version 0.97; 2004).

FIG. 18A-B show antimicrobial efficacy of IL-8_(α) in human blood andblood-derived matrices as compared with artificial media (MHB) at pH 5.5and 7.2. Panel 18 (A) shows co-incubation of IL-8_(α) and the organismsimultaneously added to the test biomatrix or medium; Panel 18 (B) showspre-incubation of IL-8_(α) in biomatrices or media for 2 h at 37° C.prior to introduction of the organism. The E. coli inocula (INOC) were10⁵ CFU/ml, and the threshold of sensitivity was considered 0.3 log 10CFU/ml.

DETAILED DESCRIPTION OF THE INVENTION

This application file contains drawings executed in color. Copies ofthis patent or application publication with color drawings will beprovided by the Office upon request and payment of the necessary fee.

This invention provides antimicrobial kinocidin peptides and relatedmethods of use. The antimicrobial kinocidin peptides of the inventionencompass at least a portion of the C-terminal α-helical region of akinocidin, wherein the C-terminal portion encompasses an α-helicalsecondary structure. The kinocidin peptides of the invention are derivedfrom and correspond to a C-terminal portion of a kinocidin that includesa γ_(KC) core predictive of antimicrobial activity. A kinocidin is aantimicrobial CXC, CX₃C, CC, or C class chemokine.

The kinocidin peptide can include up to the entire C-terminal α-helix ofthe corresponding kinocidin from which it is derived. A kinocidinpeptide generally has physicochemical properties within the ranges setforth in Table IV below and also has antimicrobial activity

The term “kinocidin,” as used herein refers to a chemokine havingmicrobicidal activity. As described herein, the ability of a chemokineto exert antimicrobial activity can be predicted based on the presenceof the γ_(KC) core consensus formula, and specific physicochemicalpatterns of amphipathicity, charge distribution, and proline positioningwithin the chemokine (see FIG. 1). More than 40 human chemokines havebeen characterized and are classified into four groups according toconserved N-terminal cysteine motifs: CXC (α-chemokines), CC(β-chemokines), C, and CX₃C (Hoffmann et al. (2002) J Leukoc Biol 72,847-855).

As used herein, the term “kinocidin peptide” refers to a peptide thathas microbicidal activity and that contains all or a portion of aC-terminal α-helix of a kinocidin. In structural terms, a kinocidinpeptide of the invention is characterized by corresponding to aC-terminal portion of a kinocidin. As described throughout thisdisclosure, a kinocidin can be selected based on the presence of theγ_(KC) core consensus formula, and specific physicochemical patterns ofamphipathicity, charge distribution, and proline positioning within thechemokine (see FIG. 12). In functional terms, a kinocidin peptide hasantimicrobial, for example, antimicrobial and/or antifungal activity,which can be confirmed via routine methods described in the art andexemplified herein. A kinocidin peptide of the invention can have anylength provided the requisite activity is present, for example, can bebetween 50 or less, 45 or less, 40 or less, 35 or less, 34 or less, 33or less, 32 or less, 30 or less, 29 or less, 28 or less, 27 or less, 26or less, 25 or less, 24 or less. 23 or less, 22 or less, 21 or less, 20or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 orless, 7 or less, 6 or less, 5 or less, and preferably between 10 and 40,more preferably between 12 and 38, more preferably between 14 and 32amino acids in length. Also encompassed within the term are dimers andother multimers, truncated molecules, and molecules that containrepetitions of particular subsequences within the motif as long as thepeptide has microbicidal activity and that contains all or a portion ofa C-terminal α-helix of a kinocidin.

The invention provides particular kinocidin peptides, each comprising aC-terminal portion of a kinocidin having an α-helical secondarystructure and antimicrobial activity, and further having a γ_(KC) core.As described herein, the kinocidin can be a CXC, CC, or C classchemokine. In one embodiment, the invention provides a kinocidin peptidethat corresponds to a CXC chemokine has the amino acid sequenceKENWVQRVVEKFLKRAENS (SEQ ID NO: 1). In another embodiment, the inventionprovides a kinocidin peptide that corresponds to a CXC chemokine has theamino acid sequence QAPLYKKIIKKLLES (SEQ ID NO: 2). In a furtherembodiment, the invention provides a kinocidin peptide that correspondsto a CXC chemokine has the amino acid sequence ASPIVKKIIEKMLNSDKSN (SEQID NO: 3). In a further embodiment, the invention provides a kinocidinpeptide that corresponds to a CXC chemokine has the amino acid sequenceDAPRIKKIVQKKLAGDES (SEQ ID NO: 4). Additional kinocidin peptides of theinvention based on Human CXC, CC and C Chemokine C-terminal α-HelicalDomains are set forth in FIG. 18.

TABLE I Antimicrobial peptides of the invention based on HumanCXC, CC and C Chemokine C-terminal α-Helical Domains. Origin NameAmino Acid Sequence (SEQ ID NO:) Group 1Based on Human CXC Chemokine α-Helical Domains CXCL1/GRO-alphaAegicidin hGro-α-C1 ASPIVKKIIEKMLNSDKSN (3) CXCL2/MIP2-alphaAegicidin hMIP2-α-C1 ASPMVKKIIEKMLKNGKSN (5) CXCL3/GRO-betaAegicidin hGro-β-C1 ASPMVQKIIEKILNKGSTN (6) CXCL4/PF-4QAPLYKKIIKKLLES (2) CXCL5/ENA-78 Aegicidin hENA-78-C1EAPFLKKVIQKILDGGNKEN (7) CXCL6/GCP-2 Aegicidin hGCP-2-C1EAPFLKKVIQKILDSGNKKN (8) CXCL7/PBP, DAPRIKKIVQKKLAGDESAD (9) CTAP3, NAP2CXCL8/IL-8 Aegicidin hIL-8-C1 KENWVQRVVEKFLKRAENS (1) CXCL9/MIGAegicidin hMIG-C1 DSADVKELIKKWEKQVSQKKKQKNGKK (359) CXCL10/IP-10Aegicidin hIP-10-C1 ESKAIKNLLKAVSKERSKRSP (10) CXCL11/I-TACAegicidin hI-TAC-C1 KSKQARLIIKKVERKNF (11) CXCL12/SDF-1Aegicidin hSDF-1-C1 KLKWIQEYLEKALNKRFKM (12) CXCL13/BCA-1Aegicidin hBCA-1-C1 QAEWIQRMMEVLRKRSSSTLPVPVFKRKIP* (13) CXCL14/BRAKAegicidin hBRAK-C1 KLQSTKRFIKWYNAWNEKRRVYEE (14) Group 2Based on Human CC Chemokine α-Helical Domains CCL1/I-309Aegicidin hI-309-C1 TVGWVQRHRKMLRHCPSKRK (15) CCL2/MCP-1Aegicidin hMCP-1-C1 KQKWVQDSMDHLDKQTQTPKT (16) CCL3/MIP-1alphaAegicidin hMIP-1α-C1 SEEWVQKYVSDLELSA (17) CCL4/MIP-1betaAegicidin hMIP-1β-C1 SESWVQEYVYDLELN (18) CCL5/RANTESAegicidin hRANTES-C1 EKKWVREYINSLEMS (19) CCL7/MCP-3 Yeaman & YountTQKWVQDFMKHLDKKTQTPKL (20) terminology: Aegicidin hMCP3-C1 CCL8/MCP-2Aegicidin hMCP-2-C1 KERWVRDSMKHLDQIFQNLKP (21) CCL11/EOTAXINAegicidin hEOTx-C1 KKKWVQDSMKYLDQKSPTPKP (22) CCL13/MCP-4Aegicidin hMCP-4-C1 KEKWVQNYMKHLGRKAHTLKT (23) CCL14/HCC-1Aegicidin hHCC-1-C1 SDKWVQDYIKDMKEN (24) CCL15/HCC-2 Aegicidin hHCC-2-C1SGPGVQDCMKKLKPYSI (25) CCL16/HCC-4 Aegicidin hHCC-4-C1NDDWVQEYIKDPNLPLLPTRNLSTVKII (26) CCL17/TARC Aegicidin hTARC-C1NNKRVKNAVKYLQSLERS (27) CCL18/PARC Aegicidin hPARC-C1NKKWVQKYISDLKLNA (28) CCL19/MIP-3beta Aegicidin hMIP-3β-C1DQPWVERIIQRLQRTSAKMKRRSS (29) CCL20/LARC Aegicidin hLARC-C1KQTWVKYIVRLLSKKVKNM (30) CCL21/SLC Aegicidin hSLC-C1KELWVQQLMQHLDKTPSPQKPAQG (31) CCL22/MDC Aegicidin hMDC-C1RVPWVKMILNKLSQ (32) CCL23/MPIF-1 Aegicidin hMPIF-1-C1SDKQVQVCVRMLKLDTRIKTRKN (33) CCL24/MPIF-2 Aegicidin hMPIF-2-C1KQEWVQRYMKNLDAKQKKASPRAR (34) CCL25/TECK Aegicidin hTECK-C1KSREVQRAMKLLDARNK* (35) CCL27/SKINKINE Aegicidin hSkine-C1QNPSLSQWFEHQERKLHGTLPKLNFGMLRKMG (36) CCL28/CCK1 Aegicidin hCCK-1-C1HNHTVKQWMKVQAAKKNGKGN* (37) Group 3Peptides Based on C Chemokine α-Helical Domains CL1/LymphotactinAegicidin hLym-C1 QATWVRDVVRSMDRKSNTRNN* (38)

Chemokines comprise a class of small secretory cytokines that playimportant roles in potentiating leukocyte chemonavigation andantimicrobial activity. More than 40 human chemokines have beencharacterized and are classified into four groups according to conservedN-terminal cysteine motifs: CXC (α-chemokines), CC (β-chemokines), C,and CX₃C (J Leukoc Biol 70, 465-466 (2001)). Chemokines have beenidentified in vertebrates as distant as teleost fish, and are expressedin a broad array of mammalian cell types including those of myeloid,endothelial, epithelial and fibroblast lineages (Hoffmann et al. (2002)J Leukoc Biol 72, 847-855). Of the chemokines, interleukin-8 (IL-8; orCXC-ligand 8 [CXCL8]) is perhaps the best characterized, having beenfirst identified as neutrophil-activating factor from human monocytesmore than 15 years ago (Walz et al. (1987) Biochem Biophys Res Commun149, 755-761; Yoshimura et al. (1987) Proc Natl Acad Sci USA 84,9233-9237).

This invention further describes methods for identifyingmultidimensional protein signatures that are useful as predictors ofprotein activity. Prior to this invention it was unknown that proteinscan be classified based on common multidimensional signatures that arepredictive of activity. While exemplified herein for a subclass ofantimicrobial peptides, this discovery allows for the invention methodsof using experimental proteomics techniques to identify multidimensionalprotein signatures that are predictive of protein activity.

Based, in part, on the discovery of structural signatures inantimicrobial peptides, the invention provides methods for designing,creating or improving anti-infective agents and anti-infectivestrategies that are refractory to microbial resistance. The inventionmethods can improve the efficacy of a drug or a drug candidate byaltering the multidimensional antimicrobial signature so as toapproximate the multidimensional signature model.

In one embodiment, the invention provides a method for predictingantimicrobial activity of a candidate protein by determining thepresence a multidimensional antimicrobial signature in a candidateprotein, and comparing the multidimensional antimicrobial signature to amultidimensional antimicrobial signature model. As taught herein, thedegree of similarity between the multidimensional antimicrobialsignature of the candidate protein and the multidimensionalantimicrobial signature model is predictive of antimicrobial activity ofthe candidate protein.

In a further embodiment, the invention provides a method for identifyinga protein having antimicrobial activity by screening a library ofcandidate proteins to identify a multidimensional antimicrobialsignature in a candidate protein, and subsequently comparing themultidimensional antimicrobial signature to a multidimensionalantimicrobial signature model. As taught herein, the degree ofsimilarity between the multidimensional antimicrobial signature of thecandidate protein and the multidimensional antimicrobial signature modelis predictive of antimicrobial activity of the candidate protein.

In a further embodiment, the invention provides a method for improvingthe antimicrobial activity of a protein by altering the multidimensionalantimicrobial signature of the protein to increase the degree ofsimilarity between the multidimensional antimicrobial signature of theprotein and a multidimensional antimicrobial signature model. Theinvention also provides a protein having improved antimicrobial activityas a result of alteration of the multidimensional antimicrobialsignature of the protein to increase the degree of similarity betweenthe multidimensional antimicrobial signature of the protein and amultidimensional antimicrobial signature model.

In a further embodiment, the invention provides a method for designing aprotein having antimicrobial activity by incorporating configurationsthat include iterations of a γ-core signature into a peptide structurethat is designed. The invention also provides a protein havingantimicrobial activity designed by incorporating configurations thatinclude iterations of a γ-core signature into a peptide structure.

As used herein, the term “multidimensional protein signature” isintended to refer to a set of essential physicochemical components thatmake up a structural motif characteristic of a class or subclass ofproteins. A multidimensional protein signature can incorporate anystructural information ascertainable, including, information regardingprimary structure, including amino acid sequence, composition, anddistribution patterns; secondary structure, stereospecific sequence and3-dimensional conformation. As used herein, the term “multidimensionalprotein signature model” refers to a protein that represents theessential structural components associated with a particularmultidimensional protein signature. Individual peptides each contain aniteration of the multidimensional signature, and the essential featuresof this signature are reflected in the multidimensional signature model.CS-αβ family antimicrobial peptides also contain a γ_(AP) core andα-helix Kinocidins, including IL-8, share a common topology comprised ofa γ_(KC) core and α-helix.

As used herein, the terms “gamma-core motif,” “γ-core,” “γ_(AP)-core,”“γ-core signature” and equivalents thereof refer to a multidimensionalprotein signature, in particular a multidimensional antimicrobialsignature, that is characterized by two anti-parallel β-sheetsinterposed by a short turn region with a conserved GXC (dextromeric) orCXG (levomeric) sequence pattern integrated into one β-sheet. Additionalfeatures that characterize the γ-core motif include a hydrophobic biastoward the C-terminal aspect and cationic charge positioned at theinflection point and termini of the β-sheet domains, polarizing chargealong the longitudinal axis of the γ-core.

The kinocidin γ-core (γ_(KC) core) signature is an iteration of theantimicrobial peptide γ-core (γ_(AP)), conforming to an anti-parallelβ-hairpin comprised of a 13-17 amino acid pattern with a centralhydrophobic region typically flanked by basic residues. The γ_(KC) coremotif can be characterized by the following consensus sequence formula:

(SEQ ID NO: 39) NH₂ [C]-[X₁₀₋₁₃]-[GX₂₋₃C]-[X₂]-[P] COOH

Human IL-8, which contains the kinocidin γ-core (γ_(KC) core) signature,has the sequence:

(SEQ ID NO: 40) NH₂ CANTEIIVKLSDGRELCLDP COOH

This fragment of the IL-8 sequence is consistent with the consensusγ_(KC)-core motif. Furthermore, many kinocidins exhibit a recurringamino acid position pattern, consistent with the consensus γ_(KC) coreformula:

(SEQ ID NO: 41) NH₂ CX₄Z₃X₀₋₂[K⁸¹]X₁₋₃G[K⁷²][B⁸⁶][Z⁹²]C[Z⁸⁶][D⁸⁶][P⁹⁵]COOH                          R             Nwhere Z represents the hydrophobic residues A, F, I, L, V, W, Y; Brepresents the charged or polar residues D, E, H, K, N, R, Q; C, P, or Gcorrespond to cysteine, proline, or glycine, respectively, X indicatesvariable amino acid position; and numeric superscripts of bracketedpositions indicate relative frequency in percent, with common alternateresidues listed beneath.

As used herein, the term “protein activity” is intended to mean afunctional activity or bioactivity of a protein.

Many disulfide-containing antimicrobial peptides have multiplestructural domains that encompass β-sheet and/or α-helical motifsconnected through an interposing region. As described herein, theinvention methods provide a strategy incorporating a synthesis ofproteomic and experimental methods to identify essential structuralfeatures integral to antimicrobial bioactivity that are shared amongstbroad classes of antimicrobial peptides. Stereospecific sequence and3-dimensional conformation analyses of cysteine-containing antimicrobialpeptides with known structures were integrated and reduced to identifyessential structural components. These approaches enabled theidentification of sequence patterns and a 3-dimensional conformationintegral to a multidimensional signature common to virtually allnon-cyclic antimicrobial peptides containing disulfide bridges. Thiscompelling signature transcends class-specific motifs identifiedpreviously, and reflects a unifying structural code in antimicrobialpeptides from organisms separated by profound evolutionary distances.

The γ-core motif is a pivotal element in the multidimensional signatureof antimicrobial peptides. This motif corresponds to a hydrophobic andstructurally rigid region in these molecules. Moreover, the γ-core motifconsists of hallmark amino acid sequence, composition, and distributionpatterns that likely facilitate antimicrobial functions. For example,patterns identified are congruent with segregation of the most polar orcharged residues to solvent-accessible facets, continuity of hydrophilicor hydrophobic surfaces, and flexibility near structural extremities ofthese peptides. Such physicochemical properties appear to be integral tothe antimicrobial mechanisms of disulfide-containing peptides such asthe CS-αβ or defensin families (Yeaman and Yount, Pharmacol. Rev. 55:27(2003); Hill et al., Science 251:1481 (1991)). Thus, the γ-core motif ismore than simply a β-hairpin fold. As described herein, the γ-corecomponent of the antimicrobial peptide signature can be derived fromdextromeric or levomeric sequence patterns (FIG. 2B). The necessity forhost defense against microbial pathogens has favored conservation of aneffective 3-dimensional determinant, despite site- ororientation-specific variations in the primary sequences that comprisethis motif. Thus, the present invention provides a method forstereospecific analysis of primary sequences that can identifystructural patterns or relationships in any protein class selected bythe user.

Conservation of the γ-core motif across the phylogenetic spectrumdemonstrates it is an archetype of the antimicrobial peptide signature(FIG. 8A). Yet, the γ-core is not necessarily an exclusive structuraldeterminant of antimicrobial activity. In some cases, the γ-core aloneis sufficient for antimicrobial activity (eg., protegrins, tachyplesins,RTD-1). However, the motif also can serve as a scaffold, to whichcomplementary antimicrobial determinants (eg., α-helices or β-sheets)are added as adjacent modules.

Thus, disulfide-stabilized antimicrobial peptides represent structuralmodules coordinated in varying configurations relative to the γ-core(FIG. 8B). Examples of the invention discovery are abundant in nature:Protegrin-1 illustrates the simplest configuration, consisting solely ofthe γ-core, represented by the modular formula [γ]; MGD-1 contains anα-helical module linked to a γ-core, collectively represented as [γ-α];alternatively, HNP-3 exemplifies the addition of a β-sheet module to theγ-core, represented as [β-γ]; Ah-AMP-1 illustrates a more complexconfiguration in which β-sheet and α-helical modules are linked to theγ-core, represented by the formula [β-γ-α]. Permutations of thesemodular formulae are readily observed in naturally-occurringantimicrobial peptides, encompassing diverse antimicrobial peptidefamilies, including α-defensins, β-defensins, θ-defensins,cathelicidins, protegrins, and CS-αβ peptides found in plants,invertebrates, insects, and arthropods. Based on this discovery thepresent invention provides methods of utilizing specific mosaicconfigurations of such structural modules to optimize the function of agiven antimicrobial peptide against relevant pathogens in specificphysiologic contexts.

Thus, peptides with common evolutionary precursors may have conservedstructural elements independent of functional divergence. As oneverification of this discovery, AFP-1 and TGF-α were intentionallyincluded in the exemplified phylogenetic and structural analyses asrelative outliers in the comparative antimicrobial and non-antimicrobialpeptide groups. This level of divergence is reflected in theirsignificant phylogenetic distances from other peptides in theirrespective subsets. Yet, as described herein, despite equidistantdivergence from Ah-AMP-1, AFP-1 exhibits the fundamental γ-coresignature of antimicrobial peptides, while TGF-α does not (FIGS. 3 I-Land 8A). This result reinforces the importance of the γ-core motif aspart of a multidimensional signature for antimicrobial activity.Moreover, structural divergence of AFP-1 from other antimicrobialpeptides lies predominantly in modules beyond the γ-core. Thus, asexemplified for AFP-1, the invention provides new insights intoeukaryotic evolution of the multidimensional signature of antimicrobialpeptides that confer survival advantages in environments rich inmicrobial pathogens.

The discovery of a multidimensional signature as described herein can beapplied to a method of identifying peptides that exert previouslyunrecognized antimicrobial activity. As described herein, for example,the sweetener protein, brazzein, and the scorpion neurotoxin,charybdotoxin, were found to have previously unrecognized antimicrobialactivity against bacteria and fungi. The present model also accuratelypredicted that the prototype metallothionein II, which fulfilled theprimary sequence pattern, but lacked the 3-dimensional criteria of theantimicrobial signature, was devoid of antimicrobial activity. Asdescribed herein, the multidimensional signature model was furthersubstantiated by successful prediction of the γ-core motif intachyplesins of unknown 3-dimensional structure, but which had knownantimicrobial activity, and fulfilled the primary structure criteria ofthe model. Together, these findings validate the predictive accuracy,utility and applicability of the multidimensional antimicrobial peptidesignature model to the methods provided by the present invention.

As disclosed herein, the multidimensional signature is a unifyingstructural code for broad classes of host defense peptides. Thisdiscovery is supported, for example, in the exemplification that a majorclass of peptides can be retrieved from the protein database searchesusing the stereospecific sequence formulae consisting of proteaseinhibitors and related proteins derived from plants (FIG. 11B). Thebotanical and related literature indicate that several such peptideshave been shown to be plant defensins (Sallenave, Biochem. Soc. Trans.30:111 (2002); Wijaya et al., Plant Sci 159:243 (2000)). Moreover, theplant proteinase inhibitor superfamily includes thionin peptidescontaining the antimicrobial γ-core motif as disclosed herein (Table I;Melo et al., Proteins 48:311 (2002)). In addition, peptides originallyidentified as having cytokine bioactivities are now known to have directantimicrobial activity. Examples include γ-chemokines such as humanplatelet factor-4 and platelet basic peptide (PF-4 and PBP; Tang et al.,Infect. Immun 70:6524 (2002); Yeaman, Clin. Infect. Dis. 25:951 (1997)),monokine induced by interferon-γ (MIG/CXCL9; Cole et al., J. Immunol.167:623 (2001)), interferon-γ inducible protein-10 kDa (IP-10/CXCL10;Cole et al., J. Immunol. 167:623 (2001)), interferon-inducible T cell achemoattractant (ITAC/CXCL11; Cole et al., J. Immunol. 167:623 (2001)),and the β-chemokine, RANTES (releasable upon activation normal T cellexpressed/secreted; Tang et al., Infect. Immun 70:6524 (2002); Yeaman,Clin. Infect. Dis. 25:951 (1997)). Importantly, each of these proteinscontains an iteration of the multidimensional antimicrobial signature asprovided by the present invention. Collectively, these observationsdemonstrate the link between the multidimensional antimicrobialsignature, and functional correlates in multifunctional host defensepeptides (Yeaman, Clin. Infect. Dis. 25:951 (1997); Ganz, Science298:977 (2002)). The skilled person will appreciate that themultidimensional antimicrobial signature can be found in additionalpeptides, and that the presence of this signature is associated withantimicrobial activity.

Multidimensional signatures of antimicrobial peptides exemplify hownature can diverge at the level of overall amino acid sequence, yetpreserve essential primary sequence patterns and 3-dimensionaldeterminants effective in host defense. Thus, critical structures ofantimicrobial peptides from evolutionarily distant organisms such asmicrobes and plants are recapitulated in higher organisms, includinghumans. As disclosed herein, vertical and horizontal acquisition ofgenes, along with their recombination, yield mosaic iterations upon keystructural determinants, such as the γ-core motif (Bevins et al.,Genomics 31:95 (1996); Gudmundsson, et al., Proc. Natl. Acad. Sci. USA92:7085 (1995)). Selective pressures favoring this remarkable degree ofstructural conservation can include genetic selection against structuralvariants, and convergent evolution of independent ancestral templates.It follows that the γ-core signature is incorporated into a variety ofstructural mosaics (eg., [γα₁], [γβ₁], or [γ₁β₁]) readily observedamongst disulfide-stabilized antimicrobial peptides along thephylogenetic spectrum. While future studies will resolve their precisephylogenetic lineage, the multidimensional signatures in antimicrobialpeptides likely reflect fundamental host-pathogen interactions and theirco-evolution.

The discovery and characterization of antimicrobial peptide signaturescan also provide insights for development of new generationanti-infective agents. For example, most microbial pathogens are unableto acquire rapid or high-level resistance to antimicrobial peptides.Critical structure-activity relationships in these molecules cancircumvent microbial resistance mechanisms, and interfere with essentialmicrobial targets distinct from classical antibiotics (Yeaman and Yount,Pharmacol. Rev. 55:27 (2003)). Such modes of action exploitpathogen-specific structures intrinsically difficult to mutate, limitingthe development of resistance through target or pathway modification.Thus, structural signatures in antimicrobial peptides can advance thediscovery and development of improved anti-infective agents andstrategies that are refractory to microbial resistance. Therefore, theinvention provides a method of improving the antimicrobial activity of aprotein by altering the multidimensional signature. Methods of proteindesign are well known in the art as described, for example, in Conceptsin Protein Engineering and Design: An Introduction; Wrede and Schneider(Eds.), Walter de Gruyter, Inc. (pub.), 1994); Evolutionary Approachesto Protein Design, Vol. 55, Frances H. Arnold (Ed.), Edward M. Scolnick(Ed.), Elsevier Science & Technology Books, 2000; Molecular Design andModeling: Concepts and Applications, Part A: Proteins, Peptides, andEnzymes: Volume 202: Molecular Design and Modelling Part A, John N.Abelson (Ed.), John J. Langone (Ed.), Melvin I. Simon (Ed.), ElsevierScience & Technology Books, 1991; and Protein Engineering and Design,Paul R. Carey (Ed.), Elsevier Science & Technology Books, 1996; all ofwhich are incorporated herein by reference in their entirety.

While chemokines have not traditionally been ascribed with directantimicrobial activities, evidence for such functions is mounting. Asdescribed above, peptides originally identified as having cytokinebioactivities, including chemokines platelet factor-4 (PF-4) plateletbasic peptide (PBP) and its derivative CTAP-3 (Tang et al. (2002) InfectImmun 70, 6524-33; Yeaman et al. (1997) Infect Immun 65, 1023-31; Yountet al. (2004) Antimicrob Agents Chemother 48, 4395-404), as well astruncations thereof (Krijgsveld et al. (2000) J Biol Chem 275,20374-81), are now known to have direct antimicrobial activity. Directantimicrobial activity was subsequently reported for other chemokines(Cole et al. (2001) J Immunol 167, 623-7; Yang et al. (2003) J LeukocBiol 74, 448-55). Hence, the term kinocidin (kino-action;cidin-microbicidal) has been applied to chemokines that also exertdirect microbicidal activity (Yount & Yeaman (2004) Proc Natl Acad SciUSA 101, 7363-8; Yount et al. (2004) Antimicrob Agents Chemother 48,4395-404, Yeaman, M. R. & Yount, N. (2005) ASM News 71, 21-27).

Despite immunological likeness with other kinocidins, priorinvestigations have not detected direct antimicrobial activity of IL-8.As described herein, primary sequence and conformation analysesspecified IL-8 and kinocidin iterations of the γ-core signature presentin broad classes of antimicrobial peptides (Yount & Yeaman (2004) ProcNatl Acad Sci USA 101). Based on these structural parallels, IL-8 wasdiscovered to have direct antimicrobial activity. Multiple lines ofinvestigation described herein confirmed that IL-8 exerts significant,context-specific antimicrobial activity, with potent efficacy againstCandida albicans. Moreover, the invention provides a synthetic congenercorresponding to the α-helical domain of IL-8 that exerts antimicrobialactivity equivalent to or exceeding that of native IL-8.

As disclosed herein, IL-8 and other kinocidins share key properties withclassical antimicrobial peptides. For example, kinocidins exhibit globalas well as local amphipathic domains, and have pI values of 8.5 orgreater, indicating net positive charge at neutral pH (Table III).However, distribution of charge within kinocidins is not uniform;typically, cationic charge is associated with the C-terminal α-helix,and termini of the IL-8γ core. Molecular modeling has suggested theseregions form electropositive facets of varying size in distinctkinocidins (Yang et al. (2003) J Leukoc Biol 74, 448-55). It is notablethat, while study kinocidins share such cationic properties, neither netcharge pI directly correlated with antimicrobial activity. For example,although IL-8 was one of the least cationic kinocidins studied (pI=9.0),it had greater efficacy than many of its more positively chargedcounterparts.

Recognition that charge alone does not account for antimicrobialactivity emphasizes the importance of 3-D structure for kinocidinfunction. Notably, the strongly anti-fungal kinocidin IL-8 bears astriking structural resemblance to classical antifungal CS-αβantimicrobial peptides of plants and insects (FIG. 13; Yount & Yeaman(2004) Proc Natl Acad Sci USA 101). Kinocidins including IL-8 share acommon topology comprised of a γ_(KC) core and α-helix. Likewise, CS-αβfamily antimicrobial peptides also contain a γ_(AP) core and α-helix(Yang et al. (2003) J Leukoc Biol 74, 448-55). However, as kinocidinsare larger (8-14 kDa) than many classical antimicrobial peptides (<5kDa), it follows that global physicochemical properties do not strictlycorrelate with antimicrobial efficacy. Rather, it is more likely thatdiscrete domains likely confer microbicidal versus chemotacticactivities for kinocidins or chemotactic antimicrobial peptides (Yeaman& Yount (2005) ASM News 71, 21-27).

The fact that the C-terminal IL-8α peptide recapitulated themicrobicidal efficacy and spectrum of native IL-8 substantiates thehypothesis that kinocidin antimicrobial effects can be mediated byspecific or even autonomous structural domains. Inspection of the IL-8αdomain revealed a highly structured helix, with physiochemical featureslikely attributable to its direct antimicrobial efficacy. Moreover, thecurrent structure analyses concur with independent NMR studies (Clore etal. (1990) Biochemistry 29, 1689-96) of the IL-8 α-helix domain. Inaddition, as its helical conformation is stable at pH 5.5 and 7.5,pH-specific antimicrobial efficacy of IL-8α relies on parameters otherthan conformation (FIG. 16). Superimposed upon the 19-residue IL-8αdomain are cationic charge and amphipathicity (FIG. 17). Its estimatedpI of 10 and net charge of +2 (at pH 7) indicate a degree of cationicpotential greater than that of corresponding domains in other studykinocidins except lymphotactin. Furthermore, its amphipathicity (6.70;Table 3) is also relatively high, as hydrophobic moments >3.0 areconsidered significant in terms of hydrophobic versus hydrophilic aminoacid segregation. Such characteristics parallel those inwell-characterized helical antimicrobial peptides (Wieprecht et al.(1997) Biochemistry 36, 6124-32; Uematsu & Matsuzaki. (2000) Biophys J79, 2075-83).

Analyses of the physicochemical properties reveal that kinocidinmolecules share key properties with classical antimicrobial peptides.For example, kinocidins exhibit global as well as local amphipathicdomains, and have pI values of 8.5 or greater, indicating net positivecharge at neutral pH (Table III). However, distribution of charge withinkinocidins is not uniform; typically, cationic charge is associated withthe C-terminal α-helix, and termini of the IL-8γ core. Molecularmodeling has suggested these regions form electropositive facets ofvarying size in distinct kinocidins (Yang et al. (2003) J Leukoc Biol74, 448-55).

The current results demonstrate direct antimicrobial activities of theseand other kinocidins against bacteria in solid-phase and solution-phaseassays, including striking fungicidal activity versus C. albicans. Thesurprising discovery of IL-8 antimicrobial efficacy may be due to anumber of factors, including organism and/or strain differences, as wellas buffer composition and pH. Prior studies using a radial diffusionassay measured activity against different organisms (Escherichia coli,Listeria monocytogenes), and did not assess activity at pH 5.5 (11). Inthe one prior study using a solution-phase assay, activity of IL-8 wasevaluated against E. coli and S. aureus using a phosphate-based buffersystem at pH 7.4 (Yang et al. (2003) J Leukoc Biol 74, 448-55). Resultsfrom these latter experiments corroborate the present finding thatnative IL-8 lacks activity against S. typhimurium or S. aureus insolution phase at pH 7.5.

The current investigations demonstrated that IL-8 exerted significantmicrobicidal efficacy at concentrations descending to the high nM range.While such concentrations reflect relatively strong microbicidalefficacy, it could be argued that even μg/ml levels of activity havelimited physiologic relevance. However, several considerations supportthe concept that the antimicrobial effects of kinocidins including IL-8observed in vitro are relevant to host defense in vivo. In normal humanplasma, IL-8 is present at a very low baseline level in the range ofpicograms/ml (30). However, in contexts of infection, circulating IL-8levels rise rapidly and dramatically as much as 1000-fold, yieldingconcentrations of 30-50 ng/ml (Moller et al. (2005) J Infect Dis 191,768-75). In the current report, IL-8 was active in the 5000-1000 ng/mlrange, 100-fold greater than the highest measured concentrations inplasma. Yet, the potential for IL-8 and other kinocidins to reachefficacious concentrations in local contexts of infection is supportedby considerable evidence. For example, recent studies by Qiu et al. showthat the chemokine CCL22/MDC reaches μg/g levels in lung granulomae (Qiuet al. (2001) Am J Pathol 158, 1503-15). Additionally, as kinocidinsadhere readily to pathogens, measurements of their free concentrationdiluted in media or sera almost certainly underestimate their localintensification (Mezzano et al. (1992) Nephron 61, 58-63). Also, thesystemic administration of α-helical antimicrobial peptides do notpreclude their concentration specifically at sites of infection(Nibbering et al. (2004) J Nucl Med 45, 321-6), perhaps by affinity ofthe cationic peptide for electronegative bacterial cell membranes. Suchevents likely achieve local concentrations of IL-8 and other kinocidinssufficient for microbicidal potency and chemotactic navigation.

In many contexts of infection or inflammation, pH of interstitialfluids, abscess exudates, and serum is significantly lower than that ofplasma. Furthermore, recurring host-defense strategies include mildacidification of mucosal epithelia and the neutrophil phagolysosome.Thus, assessment of IL-8 and subdomain antimicrobial efficacy at pH 7.5versus 5.5 was designed to reflect such microenvironments. The fact thatkinocidins, including IL-8 and the IL-8α antimicrobial domain, exertenhanced antimicrobial efficacy at pH 5.5 is consistent with theseconcepts. Thus, beyond providing a chemical barrier, such pH modulationmay contribute to mucosal surfaces that are inhospitable to microbialcolonization. A parallel line of reasoning also supports the conceptthat kinocidins mutually potentiate the antimicrobial mechanisms ofleukocytes. Kinocidins are known to interact with leukocytes viachemokine motifs, and with microorganisms via charge-mediated properties(Yang et al. (2003) J Leukoc Biol 74, 448-55). Thus, pathogenspre-decorated with kinocidins or antimicrobial domains thereof arebelieved to be more efficiently killed when internalized into the acidicphagolysosome of professional phagocytes (5). Additional support forthis concept is exemplified by studies demonstrating significantquantities of the kinocidin PBP in the phagolysosomes of activatedmacrophages (34). In these ways, kinocidins are likely evolved tofunction in specific contexts to optimize antimicrobial defenses withoutconcomitant host toxicity.

Peptides useful as antifungal or antibacterial agents are those whichare at least 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or moreamino acids in length and which comprise at least a portion of thealpha-helical structure sufficient for antimicrobial activity, andsubstitutions thereof. An example of a permissible substitutions isalanine for cysteine.

The peptides of the present invention can be chemically synthesized.Thus polypeptides can be prepared by solid phase peptide synthesis, forexample as described by Merrifield. The synthesis is typically carriedout with amino acids that are protected at the alpha-amino terminus.Trifunctional amino acids with labile side-chains are also protectedwith suitable groups to prevent undesired chemical reactions fromoccurring during the assembly of the polypeptides. The alpha-aminoprotecting group is selectively removed to allow subsequent reaction totake place at the amino-terminus. The conditions for the removal of thealpha-amino protecting group do not remove the side-chain protectinggroups.

The alpha-amino protecting groups are well known to those skilled in theart and include acyl type protecting groups (e.g., formyl,trifluoroacetyl, acetyl), aryl type protecting groups (e.g., biotinyl),aromatic urethane type protecting groups [e.g., benzyloxycarbonyl (Cbz),substituted benzyloxycarbonyl and 9-fluorenylmethyloxy-carbonyl (Fmoc)],aliphatic urethane protecting groups [e.g., t-butyloxycarbonyl (tBoc),isopropyloxycarbonyl, cyclohexloxycarbonyl] and alkyl type protectinggroups (e.g., benzyl, triphenylmethyl). The preferred protecting groupsare tBoc and Fmoc.

The side-chain protecting groups selected must remain intact duringcoupling and not be removed during the deprotection of theamino-terminus protecting group or during coupling conditions. Theside-chain protecting groups are also removable upon the completion ofsynthesis using reaction conditions that will not alter the finishedpolypeptide. In tBoc chemistry, the side-chain protecting groups fortrifunctional amino acids are mostly benzyl based. In Fmoc chemistry,they are mostly tert-butyl or trityl based.

In addition, the peptides can also be prepared by recombinant DNAtechnologies wherein host cells are transformed with proper recombinantplasmids containing the nucleotide sequence encoding the particularpeptide. The peptides of the present invention can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryoticcells, particularly cultured cells of multicellular organisms, arepreferred. Techniques for manipulating cloned DNA molecules andintroducing exogenous DNA into a variety of host cells are well known inthe art and can be found in standard references as Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001) and Ansubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999), both ofwhich are incorporated herein by reference.

In general, a DNA sequence encoding a peptide of the present inventionis operably linked to other genetic elements required for itsexpression, generally including a transcription promoter and terminatorwithin an expression vector. The vector typically contains one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are available throughcommercial suppliers.

The peptides of the present invention can be formulated intocompositions in pharmaceutically acceptable carriers for administrationto individuals. For oral administration, the peptides can be formulatedinto a solid preparation such as tablets, pills, granules, powder,capsules and the like, or a liquid preparation such as solutions,suspensions, emulsions and the like. The pharmaceutical preparations fororal administration comprising one or more peptides of the presentinvention may also contain one or more of the following customaryexcipients: fillers and extenders including starches, lactose, sucrose,glucose, mannitol and silica; binders including carboxymethylcellulose,alginates, gelatine and polyvinylpyrrolidone; humectants includingglycerine; disintegrating agents, including agar-agar, calcium carbonateand sodium carbonate; solution retarders, including paraffin; absorptionaccelerators including quaternary ammonium compound; wetting agentsincluding cetyl alcohol or glycerine monostearate; adsorbents includingkaolin and bentonite; lubricants including talc, calcium stearate andmagnesium stearate and solid polyethylene glycols; colorants;flavorings; and sweeteners.

When the preparation is used for parental administration, thepreparation is made in an injection formula. For the preparation of aninjection formula, the solutions and emulsions can be in a sterile formwhich is isotonic with blood. The suspensions can contain in addition tothe active peptide or peptides, preservatives, stabilizers,solubilizers, wetting agents, salts for changing the osmotic pressure orbuffers.

The peptides of the present invention are useful as antifungal orantibacterial agents.

The invention provides methods of using kinocidin peptide constructssuch as IL-8α for treating a subject suffering from infection (includingfungal, bacterial, or other microbial infection), especially mammaliansubjects such as humans, but also including farm animals such as cows,sheep, pigs, horses, goats and/or poultry (e.g., chickens, turkeys,ducks and/or geese), companion animals such as dogs and/or cats, exoticand/or zoo animals, and/or laboratory animals including mice, rats,rabbits, guinea pigs, and/or hamsters. Immunocompromised orimmunosuppressed subjects, e.g., subjects suffering from cancer,subjects undergoing radiation therapy and/or cytotoxic chemotherapy,subjects being treated with immunosuppressive drugs, and/or subjectssuffering from natural or acquired immune deficiencies such as AIDS, maybe treated according to this aspect of the invention. Treatment ofinfection of plants is also contemplated.

“Treatment” as used herein encompasses both prophylactic and/ortherapeutic treatment, and may be accompanied by concurrentadministration of other antimicrobial agents, including any of theagents discussed herein.

Fungal infection that may be treated according to the invention may becaused by a variety of fungal species including Candida (including C.albicans, C. tropicalis, C. parapsilosis, C. stellatoidea, C. krusei, C.parakrusei, C. lusitanae, C. pseudotropicalis, C. guilliermondi, C.dubliniensis, C. famata or C. glabrata), Aspergillus (including A.fumigatus, A. flavus, A. niger, A. nidulans, A. terreus, A. sydowii, A.flavatus, or A. glaucus), Cryptococcus, Histoplasma, Coccidioides,Paracoccidioides, Blastomyces, Basidiobolus, Conidiobolus, Rhizopus,Rhizomucor, Mucor, Absidia, Mortierella, Cunninghamella, Saksenaea,Pseudallescheria, Paecilomyces, Fusarium, Trichophyton, Trichosporon,Microsporum, Epidermophyton, Scytalidium, Malassezia, Actinomycetes,Sporothrix, Penicillium, Saccharomyces or Pneumocystis.

Other infections that may be treated using a peptide construct accordingto the invention may be caused by gram-negative bacterial species thatinclude Acidaminococcus, Acinetobacter, Aeromonas, Alcaligenes,Bacteroides, Bordetella, Branhamella, Brucella, Burkholderia,Calymmatobacterium, Campylobacter, Cardiobacterium, Chromobacterium,Citrobacter, Edwardsiella, Enterobacter, Escherichia, Flavobacterium,Francisella, Fusobacterium, Haemophilus, Klebsiella, Legionella,Moraxella, Morganella, Neisseria, Pasturella, Plesiomonas,Porphyromonas, Prevotella, Proteus, Providencia, Pseudomonas,Salmonella, Serratia, Shigella, Stentrophomonas, Streptobacillus,Treponema, Veillonella, Vibrio, or Yersinia species; Chlamydia; orgram-positive bacterial species that include Staphylococcus,Streptococcus, Micrococcus, Peptococcus, Peptostreptococcus,Enterococcus, Bacillus, Clostridium, Lactobacillus, Listeria,Erysipelothrix, Propionibacterium, Eubacterium, Nocardia, Actinomyces,or Corynebacterium species as well as Mycoplasma, Ureaplasma, orMycobacteria.

Other infections include infections by protozoa including Plasmodia,Toxoplasma, Leishmania, Trypanosoma, Giardia, Entamoeba, Acanthamoeba,Nagleria, Hartmanella, Balantidium, Babesia, Cryptosporidium, Isospora,Microsporidium, Trichomonas or Pneumocystis species; or infections byother parasites include helminths.

Other therapeutic uses of kinocidin peptide constructs such as IL-8αaccording to the invention include methods of treating conditionsassociated with endotoxin, such as exposure to gram-negative bacterialendotoxin in circulation, endotoxemia, bacterial and/orendotoxin-related shock and one or more conditions associated therewith,including a systemic inflammatory response, cytokine overstimulation,complement activation, disseminated intravascular coagulation, increasedvascular permeability, anemia, thrombocytopenia, leukopenia, pulmonaryedema, adult respiratory distress syndrome, renal insufficiency andfailure, hypotension, fever, tachycardia, tachypnea, and metabolicacidosis. Thus, not only gram-negative bacterial infection but alsoconditions which are associated with exposure to gram-negative bacterialendotoxin (infection-related conditions) may be ameliorated throughendotoxin-binding or endotoxin-neutralizing activities of kinocidinpeptide constructs such as IL-8α.

Therapeutic compositions of the peptide construct may include apharmaceutically acceptable diluent, adjuvant, or carrier. The peptideconstruct may be administered without or in conjunction with knownsurfactants, other chemotherapeutic agents or additional knownantimicrobial agents.

Compositions, including therapeutic compositions, of the peptideconstruct of the invention may be administered systemically ortopically. Systemic routes of administration include oral, intravenous,intramuscular or subcutaneous injection (including into depots forlong-term release), intraocular or retrobulbar, intrathecal,intraperitoneal (e.g. by intraperitoneal lavage), intrapulmonary (usingpowdered drug, or an aerosolized or nebulized drug solution), ortransdermal. Topical routes include administration in the form ofrinses, washes, salves, creams, jellies, drops or ointments (includingopthalmic and otic preparations), suppositories, such as vaginalsuppositories, or irrigation fluids (for, e.g., irrigation of wounds).

Suitable dosages include doses ranging from 1 mg/kg to 100 mg/kg per dayand doses ranging from 0.1 mg/kg to 20 mg/kg per day. When givenparenterally, compositions are generally injected in one or more dosesranging from 1 mg/kg to 100 mg/kg per day, preferably at doses rangingfrom 0.1 mg/kg to 20 mg/kg per day, and more preferably at doses rangingfrom 1 to 20 mg/kg/day. As described herein for compositions with IL-8α,parenteral doses of 0.5 to 5 mg/kg/day are preferred according to thepresent invention. The treatment may continue by continuous infusion orintermittent injection or infusion, or a combination thereof, at thesame, reduced or increased dose per day for as long as determined by thetreating physician. An antimicrobial composition can be effective atblood serum concentrations as low as 1 μg/ml. When given topically,compositions are generally applied in unit doses ranging from 1 mg/mL to1 gm/mL, and preferably in doses ranging from 1 mg/mL to 100 mg/mL.Decontaminating doses are applied including, for example, for fluids orsurfaces or to decontaminate or sterilize surgical or other medicalequipment or implantable devices, including, for example prostheticjoints or in indwelling invasive devices. Those skilled in the art canreadily optimize effective dosages and administration regimens fortherapeutic, including decontaminating, compositions as determined bygood medical practice and the clinical condition of the individualsubject.

“Concurrent administration,” or “co-administration,” as used hereinincludes administration of one or more agents, in conjunction orcombination, together, or before or after each other. The agents may beadministered by the same or by different routes. If administered via thesame route, the agents may be given simultaneously or sequentially, aslong as they are given in a manner sufficient to allow all agents toachieve effective concentrations at the site of action. For example, apeptide construct may be administered intravenously while the secondagent(s) is(are) administered intravenously, intramuscularly,subcutaneously, orally or intraperitoneally. A peptide construct and asecond agent(s) may be given sequentially in the same intravenous lineor may be given in different intravenous lines. Alternatively, a peptideconstruct may be administered in a special form for gastric or aerosoldelivery, while the second agent(s) is(are) administered, e.g., orally.

Concurrent administration of the peptide construct of the invention,such as IL-8α, for adjunctive therapy with one or more otherantimicrobial agents (particularly antifungal agents) is expected toimprove the therapeutic effectiveness of the antimicrobial agents. Thismay occur through reducing the concentration of antimicrobial agentrequired to eradicate or inhibit target cell growth, e.g., replication.Because the use of some antimicrobial agents is limited by theirsystemic toxicity, lowering the concentration of antimicrobial agentrequired for therapeutic effectiveness reduces toxicity and allows wideruse of the agent. For example, concurrent administration of the peptideconstruct, such as IL-8α, and another antifungal agent may produce amore rapid or complete fungicidal or fungistatic effect than could beachieved with either agent alone. Administration of the peptideconstruct, such as IL-8α, may reverse the resistance of fungi toantifungal agents or may convert a fungistatic agent into a fungicidalagent. Similar results may be observed upon concurrent administration ofthe peptide construct, such as IL-8α, with other antimicrobial agents,including antibacterial and/or anti-endotoxin agents.

Therapeutic effectiveness in vivo is based on a successful clinicaloutcome, and does not require that the antimicrobial agent or agentskill 100% of the organisms involved in the infection. Success depends onachieving a level of antimicrobial activity at the site of infectionthat is sufficient to inhibit growth or replication of the pathogenicorganism in a manner that tips the balance in favor of the host. Whenhost defenses are maximally effective, the antimicrobial effect requiredmay be minimal. Reducing organism load by even one log (a factor of 10)may permit the host's own defenses to control the infection. Inaddition, augmenting an early microbicidal/microbistatic effect can bemore important than a long-term effect. These early events are asignificant and critical part of therapeutic success, because they allowtime for host defense mechanisms to activate.

In addition, the invention provides a method of killing or inhibitinggrowth of pathogenic organisms (particularly fungi) comprisingcontacting the organism with the peptide construct, such as IL-8α,optionally in conjunction with other antimicrobial agents. This methodcan be practiced in vivo, ex vivo, or in a variety of in vitro uses suchas to decontaminate fluids or surfaces or to sterilize surgical or othermedical equipment or implantable devices, including prosthesesorintrauterine devices. These methods can also be used for in situdecontamination and/or sterilization of indwelling invasive devices suchas intravenous lines and catheters, which are often foci of infection.

A further aspect of the invention involves use of the peptide construct,such as IL-8α, for the manufacture of a medicament for treatment ofmicrobial infection (e.g., fungal or bacterial infection) or amedicament for concurrent administration with another agent fortreatment of microbial infection. The medicament may optionally comprisea pharmaceutically acceptable diluent, adjuvant or carrier and also mayinclude, in addition to the kinocidin peptide construct, otherchemotherapeutic agents.

Known antifungal agents which can be co-administered or combined withthe kinocidin peptide construct according to the invention includepolyene derivatives, such as amphotericin B (including lipid orliposomal formulations thereof) or the structurally related compoundsnystatin or pimaricin; flucytosine (5-fluorocytosine); azole derivatives(including ketoconazole, clotrimazole, miconazole, econazole,butoconazole, oxiconazole, sulconazole, tioconazole, terconazole,fluconazole, itraconazole, voriconazole [Pfizer], posaconazole[SCH56592, Schering-Plough]) or ravuconazole [Bristol-Myers Squibb];allylamines-thiocarbamates (including tolnaftate, naftifine orterbinafine); griseofulvin; ciclopirox; haloprogin; echinocandins(including caspofungin [MK-0991, Merck], FK463 [Fujisawa], cilofungin[Eli Lilly] or VER-002 [Versicor]); nikkomycins; or sordarins.

The polyene derivatives, which include amphotericin B or thestructurally related compounds nystatin or pimaricin, are broad-spectrumantifungals that bind to ergosterol, a component of fungal cellmembranes, and thereby disrupt the membranes. Amphotericin B is usuallyeffective for systemic mycoses, but its administration is limited bytoxic effects that include fever, kidney damage, or other accompanyingside effects such as anemia, low blood pressure, headache, nausea,vomiting or phlebitis. The unrelated antifungal agent flucytosine(5-fluorocytosine), an orally absorbed drug, is frequently used as anadjunct to amphotericin B treatment for some forms of candidiasis orcryptococcal meningitis. Its adverse effects include bone marrowdepression, including with leukopenia or thrombocytopenia.

Known antibacterial agents which can be co-administered or combined withthe peptide construct according to the invention include antibiotics,which are natural chemical substances of relatively low molecular weightproduced by various species of microorganisms, such as bacteria(including Bacillus species), actinomycetes (including Streptomyces) orfungi, that inhibit growth of or destroy other microorganisms.Substances of similar structure and mode of action may be synthesizedchemically, or natural compounds may be modified to producesemi-synthetic antibiotics. These biosynthetic and semi-syntheticderivatives are also effective as antibiotics. The major classes ofantibiotics include (1) the β-lactams, including the penicillins,cephalosporins or monobactams, including those with β-lactamaseinhibitors; (2) the aminoglycosides, e.g., gentamicin, tobramycin,netilmycin, or amikacin; (3) the tetracyclines; (4) the sulfonamidesand/or trimethoprim; (5) the quinolones or fluoroquinolones, e.g.,ciprofloxacin, norfloxacin, ofloxacin, moxifloxacin, trovafloxacin,grepafloxacin, levofloxacin or gatifloxacin (6) vancomycin; (7) themacrolides, which include for example, erythromycin, azithromycin, orclarithromycin; or (8) other antibiotics, e.g., the polymyxins,chloramphenicol, rifampin, the lincosamides, or the oxazolidinones.

Some drugs, for example, aminoglycosides, have a small therapeuticwindow. For example, 2 to 4 μg/ml of gentamicin or tobramycin may berequired for inhibition of bacterial growth, but peak concentrations inplasma above 6 to 10 μg/ml may result in ototoxicity or nephrotoxicity.These agents are more difficult to administer because the ratio of toxicto therapeutic concentrations is very low. Antimicrobial agents thathave toxic effects on the kidneys and that are also eliminated primarilyby the kidneys, such as the aminoglycosides or vancomycin, requireparticular caution because reduced elimination can lead to increasedplasma concentrations, which in turn may cause increased toxicity. Dosesof antimicrobial agents that are eliminated by the kidneys must bereduced in patients with impaired renal function. Similarly, dosages ofdrugs that are metabolized or excreted by the liver, such aserythromycin, chloramphenicol, or clindamycin, must be reduced inpatients with decreased hepatic function. In situations where anantimicrobial agent causes toxic effects, the kinocidin peptideconstruct, such as IL-8α, can act to reduce the amount of thisantimicrobial agent needed to provide the desired clinical effect.

The susceptibility of a bacterial species to an antibiotic is generallydetermined by any art recognized microbiological method. A rapid butcrude procedure uses commercially available filter paper disks that havebeen impregnated with a specific quantity of the antibiotic drug. Thesedisks are placed on the surface of agar plates that have been streakedwith a culture of the organism being tested, and the plates are observedfor zones of growth inhibition. A more accurate technique, the brothdilution susceptibility test, involves preparing test tubes containingserial dilutions of the drug in liquid culture media, then inoculatingthe organism being tested into the tubes. The lowest concentration ofdrug that inhibits growth of the bacteria after a suitable period ofincubation is reported as the minimum inhibitory concentration.

The resistance or susceptibility of an organism to an antibiotic isdetermined on the basis of clinical outcome, i.e., whetheradministration of that antibiotic to a subject infected by that organismwill successfully cure the subject. While an organism may literally besusceptible to a high concentration of an antibiotic in vitro, theorganism may in fact be resistant to that antibiotic at physiologicallyrealistic concentrations. If the concentration of drug required toinhibit growth of or kill the organism is greater than the concentrationthat can safely be achieved without toxicity to the subject, themicroorganism is considered to be resistant to the antibiotic. Tofacilitate the identification of antibiotic resistance or susceptibilityusing in vitro test results, the National Committee for ClinicalLaboratory Standards (NCCLS) has formulated standards for antibioticsusceptibility that correlate clinical outcome to in vitrodeterminations of the minimum inhibitory concentration of antibiotic.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative examples.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Identification of Multidimensional Signatures of AntimicrobialPeptides

This Example shows identification of a disulfide-stabilized core motifthat is integral to the 3-dimensional signature of cysteine-containingantimicrobial peptides.

The relatedness amongst primary structures was examined in prototypiccysteine-containing antimicrobial peptide sequences representing taxaspanning an evolutionary distance of 2.6 billion years (BY; estimateddate of phylogenetic divergence of fungi and plants from higherorganisms; Nei et al., Proc. Natl. Acad. Sci. USA. 98:2497 (2001)). Aprototype from each class of non-cyclic, disulfide-containingantimicrobial peptides was represented in these analyses [Antimicrobialpeptides were selected from the National Center for BiotechnologyInformation (NCBI) Entrez Protein (www.ncbi.nlm nih.gov:80/entrez/) orAntimicrobial Sequences (www.bbcm.univ.trieste.it/˜tossi/) databases.]

The specific criteria for selection of peptides analyzed included: 1)eukaryotic origin; 2) published antimicrobial activity; 3) non-enzymaticmechanism(s) of action; 4) mature protein sequence; and 5) less than 75amino acids in length. Peptides for which structures have beendetermined were used in structural analyses. [Peptides were selectedfrom the National Center for Biotechnology Information (NCBI) structure(www.ncbi.nlm nih.gov:80/entrez/) and Protein Data Bank (PDB)(www.rcsb.org/pdb/) resources.] The resulting study set includedantimicrobial peptides encompassing a broad distribution in source(i.e., biological kingdoms ranging from microorganisms to man), aminoacid sequence, and conformation class (FIG. 1) Amino acid sequence datawere used for these analyses, as not all nucleotide sequences have beencharacterized, and saturation of nucleotide sequence data occurs withinnon-mitochondrial sequences over evolutionary timescales.

FIG. 1 shows conventional antimicrobial peptide structure classificationand distribution. The relationship amongst structure and predominance issummarized for the commonly recognized antimicrobial peptide classes.Concatenation represents the proportionate distribution of peptidesencompassing a given structural class, as calculated from theAntimicrobial Sequences Database. Antimicrobial peptides were selectedfrom the National Center for Biotechnology Information (NCBI) EntrezProtein (www.ncbi.nlm nih.gov:80/entrez/) or Antimicrobial Sequences(www.bbcm.univ.trieste.it/˜tossi/) databases. The numbers of peptidesclassified in each group are indicated in brackets for each class. Ofthe more than 750 peptides present in the database at the onset of thestudy, the balance of those not indicated are comprised of peptidesrepresenting unusual or other classifications, including macrocyclic,proline-rich, tryptophan-rich or indolicidin-like peptides, and largepolypeptides greater than 75 amino acids in length.

Representatives included antimicrobial peptides from taxa encompassingbroad biological diversity spanning an evolutionary distance of 2.6billion years (estimated divergence of fungi and plants from higherorganisms; [Nei et al, Proc. Natl. Acad. Sci. USA 98:2497 (2001).]).This dataset included prototypes of all major classes ofdisulfide-containing antimicrobial peptides, including distinctconformation groups such as defensin, cysteine-stabilized αβ, ranaboxand β-hairpin.

Conventional MSA (N to C terminal; dextromeric) revealed no clearconsensus patterns amongst primary sequences of the antimicrobialpeptide study set. However, visual inspection revealed an absolutelyconserved GXC motif, oriented in reverse in some peptides. Wehypothesized that conventional MSA failed to recognize this invertedconsensus pattern. Therefore, peptides containing inverted GXC motifswere aligned in their C to N terminal (levomeric) orientation. Thisstereospecific MSA revealed a novel and striking sequence pattern commonto all disulfide-containing antimicrobial peptide classes (FIG. 2B). Theconsensus patterns, defined herein as the enantiomeric sequencesignature, adhere to the formulae:

(dextromeric isoform) (SEQ ID NO: 360)NH₂···[X₁₋₃]-[GXC]-[X₃₋₉]-[C]···COOH (levomeric isoform 1)(SEQ ID NO: 361) NH₂···[C]-[X₃₋₉]-[CXG]-[X₁₋₃]···COOH(levomeric isoform 2) (SEQ ID NO: 362)NH₂···[C]-[X₃₋₉]-[GXC]-[X₁₋₃]···COOH

These consensus patterns transcend defensin-specific motifs identifiedpreviously (White et al., Curr. Opin. Struct. Biol. 5:521 (1995); Yountet al., J. Biol. Chem. 274:26249 (1999). Specific characteristics of theenantiomeric sequence signatures include: i) a length of 8-16 amino acidresidues; and ii) conserved GXC or CXG motifs within the sequenceisoforms. Interestingly, levomeric isoform 2 peptides retain adextromeric GXC motif within the levomeric sequence signature (FIG. 2B).

Identification of the conserved enantiomeric signature suggested that acorresponding motif would also be present in the 3-dimensionalstructures of disulfide-stabilized antimicrobial peptides. Conformationalignments revealed a core motif that was absolutely conserved acrossall classes of disulfide-stabilized antimicrobial peptides (FIG. 3A-H;Table 1). This 3-dimensional archetype, termed herein as the γ-coremotif, is comprised of two anti-parallel β-sheets, interposed by a shortturn region (FIGS. 4 and 5). All three isoforms of the enantiomericsequence signature conform to the γ-core motif, reflecting their3-dimensional convergence (FIG. 5). Additional features thatcharacterize the γ-core include: 1) net cationic charge (+0.5 to +7)with basic residues typically polarized along its axis; 2) periodiccharge and hydrophobicity yielding amphipathic stereogeometry; and 3)participation in 1-4 disulfide bonds. This motif may comprise the entirepeptide, or link to adjacent structural domains.

Relative to the γ-core, disulfide-stabilized antimicrobial peptides ofevolutionarily distant organisms exhibited a striking convergence inconformation, that was essentially isomeric, or at a minimum, highlyhomologous (FIG. 3). This 3-dimensional convergence encompassed overallconformations, or localized to specific domains in comparative peptides.For example, the structures of Ah-AMP-1 (horsechestnut tree, Aesculus)and drosomycin (fruit fly, Drosophila) are essentially superimposableover their entire backbone trajectories (FIG. 3C). Alternatively,protegrin-1 (domestic pig, Sus) and Ah-AMP-1 share conformationalhomology corresponding to their γ-core motifs (FIG. 3A). As anticipated,magainin aligned to the α-helical motif in Ah-AMP-1 (FIG. 3G), verifyingthe specificity of conformational alignments.

To confirm the significance of 3-dimensional convergence in theantimicrobial peptide signature, comparisons between representativecysteine-containing antimicrobial and non-antimicrobial peptides ofequivalent molecular weight were performed and analyzed. Outcomesemphasize that non-antimicrobial peptides fail to achieve themultidimensional signature of antimicrobial peptides (FIG. 3I-L). Meanquantitative RMSD confirmed the statistical significance of thedifferences between antimicrobial and non-antimicrobial structures(Table II).

TABLE II Quantitative analysis of 3-dimensional convergence amongstprototypic antimicrobial peptide structures. AAs RMSD (Å) Identity (%)Align/Gap Antimicrobial Peptides Ah-AMP-1 (Aesculus; Tree; 1BK8;Wieprecht 50 0.0 100 50/0 et al. (1997) Biochemistry 36, 6124-32)Sapecin (Sarcophaga, Fly; 1L4V; Uematsu, N. 40 0.9 25.0 38/0 &Matsuzaki, K. (2000) Biophys J 79, 2075-83) Protegrin-1 (Sus; Pig; 1PG1;Fahrner et al., 19 1.2 18.8 16/0 Chem. Biol. 3: 543 (1996)) Drosomycin(Drosophila; Fruit Fly; 1MYN; 44 1.4 29.3 41/6 Landon et al., ProteinSci. 6: 1878 (1997)) Defensin (Raphanus; Radish; 1AYJ; Fant et al., 511.3 47.6 49/0 J. Mol. Biol. 279: 257 (1998)) Thionin (Triticalis; Wheat;1GPS; Bruix et al., 47 1.8 26.1 46/3 Biochemistry 32: 715 (1993)) MGD-1(Mytilus; Mussel; 1FJN; Yang et al., 39 2.0 26.5 34/1 Biochemistry 39:14436 (2000)) Thanatin (Podisus; Soldier Bug; 8TFV; 21 2.2 12.5 16/0Mandard et al., Eur. J. Biochem. 256: 404 (1998)) HNP-3 (Homo; Human;1DFN; Hill et al., 34 3.2 8.3  24/17 Science 251: 1481 (1991)) MBD-8(Mus; Mouse; 1E4R; Bauer et al., 35 3.4 0.0  24/13 Protein Sci. 10: 2470(2001)) AFP-1 (Aspergillus; Fungus; 1AFP; Campos- 51 4.8 6.2 32/7 Olivaset al., Biochemistry 34: 3009 (1995)) Mean ± SD 2.2 ± 1.2*Non-Antimicrobial Peptides TGF-α (Homo; Human; 3TGF; Harvey et al., 504.7 3.1 32/7 Eur. J. Biochem. 198: 555 (1991)) Metallothionein(Saccharomyces; Yeast; 1AOO; 40 5.3 18.8  32/16 Peterson et al., FEBSLett. 379: 85 (1996)) Allergen-5 (Ambrosia; Ragweed; 2BBG; 40 6.5 18.832/7 Metzler et al., Biochemistry 31: 5117 (1992)) Ferredoxin(Clostridium; Bacteria; 2FDN; 55 7.4 5.0 40/6 Dauter et al.,Biochemistry 36: 16065 (1997)) Mean ± SD 6.0 ± 1.2*

Briefly, three-dimensional alignments of representative antimicrobialand control non-antimicrobial peptide structures were analyzed bypairwise comparison with Ah-AMP-1 (Aesculus; horsechestnut tree; 1BK8)using the combinatorial extension method (Shindyalov and Bourne, ProteinEng. 11:739 (1998)). Control peptides were selected from a cohort of 54appropriate comparators based on disulfide content, sequence length, andmolecular weight equivalence to Ah-AMP-1. Representative results areshown. The comparative length of each mature peptide is indicated as thenumber of amino acids (AAs). Root Mean Square Deviation (RMSD) valueswere determined for distances between α-carbon atoms over the length ofthe alignment. Percent identity is the percentage of sequence identitybetween the two peptides compared. The align/gap value indicates thenumber of residues considered for the alignment, and the number of gapsinserted. Relative gap penalties were integrated into the analysis. MeanRMSD values from antimicrobial versus non-antimicrobial peptides weresignificantly different (*) as determined by two tailed T-test (P<0.01).Information for each structure is formatted as follows: peptide name,(source genus; common name; Protein Data Bank [PDB] accession code;reference).

A highly conserved, disulfide-stabilized core motif was discovered to beintegral to the 3-dimensional signature of cysteine-containingantimicrobial peptides. This feature is termed herein as the gamma-coremotif (γ-core; FIG. 5). This structural motif is comprised of twoanti-parallel β-sheets interposed by a short turn region. Notably, asshown in FIG. 4, the sequence patterns corresponding to the γ-coresignature extends across the entire range of antimicrobial peptidefamilies. Exemplary peptides incuded within the groups are: gomesin([1KFP], Acanthoscurria, spider, (γ-Group); Mandard et al., Eur. J.Biochem. 269:1190 (2002)); protegrin-1 ([1PG1], Sus, domestic pig,(γ-Group)); thanatin ([8TFV], Podisus, soldier bug, (γ-Group));α-defensin (HNP-3, [1DFN]; Homo, human, (β-γ-Group); β-defensin (MBD-8,[1E4R], Mus, mouse, (β-γ-Group)); fungal peptide (AFP-1, [1AFP],Aspergillus, fungus, (β-γ-α Group); insect-defensin (sapecin, [1L4V],Sarcophaga, flesh fly, (γ-α-Group)); crustacean

CS-αβ peptide (MGD-1, [1FJN], Mytilus, mussel, (γ-α-Group)); insectCS-αβ peptide (drosomycin, [1MYN], Drosophila, fruit fly, (γ-α-Group));and plant CS-αβ□ peptide (Ah-AMP-1, [1BK8] Aesculus, horsechestnut tree,(β-γ-α Group)□. Other peptide data are formatted as in FIG. 3. See TableII for additional references. The conserved GXC (dextromeric) or CXG(levomeric) sequence patterns (FIG. 2B) are integrated into one (3-sheetin this motif, reflecting conformational symmetry amongst antimicrobialpeptides containing this signature (FIG. 5, respectively). Additionalfeatures that distinguish the γ-core include: 1) hydrophobic bias towardthe C-terminal aspect; and 2) cationic charge positioned at theinflection point and termini of the β-sheet domains, polarizing chargealong the longitudinal axis of the γ-core.

Example II Validation of the Multidimensional Antimicrobial PeptideSignature Model

The multidimensional signature model for antimicrobial peptidesintegrates a stereospecific (dextromeric or levomeric) sequence patternwith the 3-dimensional gamma-core (“γ-core”). Therefore, this modelpredicted that peptides fulfilling these prerequisites would exertantimicrobial activity, even though such activity may not yet have beendetermined. Multiple and complementary approaches were used to test themodel in this regard: 1) prediction of antimicrobial activity inpeptides fulfilling the sequence and conformation criteria of themultidimensional signature, but not yet recognized to have antimicrobialactivity; 2) predicted failure of antimicrobial activity in peptidesexhibiting primary sequence criteria, but lacking the 3-dimensional□γ-core signature of the model; and 3) prediction of a γ-core motif indisulfide-containing peptides with known antimicrobial activity, andwhich fulfilled primary sequence criteria, but had unknown structure.

To test the hypothesis that the primary sequence patterns of themultidimensional signature are relevant to all classes ofdisulfide-containing antimicrobial peptides, Swiss-Prot forward andreverse databases (Gattiker et al., Appl. Bioinformatics 1:107 (2002))were queried with the enantiomeric sequence formulae. Representatives ofall major disulfide-containing antimicrobial peptide classes wereretrieved (Table III). Searches also retrieved members of other peptidesubclasses: i) neurotoxins, particularly charybdotoxin class of thefamily Buthidae (scorpion); ii) protease inhibitor or related peptides(eg., brazzein) from plants; iii) ferredoxins; and iv) metallothioneins.Prototypes with known 3-dimensional structures, but no knownantimicrobial activity, were analyzed for the presence of the γ-coresignature. Of these, the peptides brazzein and charybdotoxin wereselected to test for antimicrobial activity based on two criteria: i)their quantitative RMSD values reflected greatest homology to thecomparator γ-core motif; and ii) they represented diverse non-mammalian(plant or scorpion) host sources and distinct structure classes notpreviously known to have antimicrobial activity. Thus, brazzein andcharybdotoxin exemplified peptides that fulfilled the enantiomericsequence and γ-core criteria required for the multidimensionalsignature. These peptides were predicted to have direct antimicrobialactivity. In contrast, prototype metallothioneins and ferredoxins didnot contain γ-core motifs (FIG. 3; Table II). Thus, metallothionein IIwas selected as an example comparator predicted to lack antimicrobialactivity.

TABLE III Recognition of diverse classes of antimicrobial peptides bythe enantiomeric sequence formulae. Forward or reverse Swiss-ProtDatabases (release 42.4; Nov. 14, 2003; 138,347 entries) were probedwith formulae containing the dextromeric or levomeric motifs of theantimicrobial peptide signature using PROSITE (Gattiker et al., Appl.Bioinformatics 1: 107 (2002)). Data indicate the proportionatedistribution of a non-redundant cohort of retrieval sets; in some cases,peptides were retrieved by more than one formula isoform. Note thatsearch results include members of the lantibiotic superfamily ofantimicrobial peptides that lack conventional disulfide bridges, buthave alternate thioether stabilization. Sequence Isoform ProportionAntimicrobial Peptide Class Phylogeny Dextro Levo - 1 Levo - 2 Total %Total α-defensin Chordata 24 42 6 72 15.3 β-defensin Chordata 52 65 31148 31.4 θ-defensin Chordata 1 1 0 2 0.4 Insect defensin/CS-αβ Insectae21 23 12 56 11.9 Plant defensin/CS-αβ Plantae 51 67 20 138 29.3Invertebrate defensin/CS-αβ Mollusca 3 4 4 11 2.3 Protegrins/GomesinsChordata/Arthropoda 0 0 6 6 1.3 Tachyplesins/Polyphemusins Arthropoda 65 2 13 2.8 Thanatin Arthropoda 0 1 0 1 0.2 Mytilins/Big-DefensinMollusca 3 3 2 8 1.7 AFP-1 Ascomycota 1 0 0 1 0.2 Lantibiotics/MicrocinsProteobacteria 3 3 9 15 3.2 165 214 92 471

These peptides were tested for antimicrobial activity against a panel ofGram-positive (Staphylococcus aureus, Bacillus subtilis) andGram-negative (Escherichia coli) bacteria, and the fungus Candidaalbicans, using a well-established and sensitive in vitro assay[Antimicrobial activity was assessed using a well-establishedsolid-phase diffusion method. Assays included well-characterizedorganisms: Staphylococcus aureus (ATCC 27217, Gram-positive); Bacillussubtilis (ATCC 6633, Gram-positive); Escherichia coli (strain ML-35,Gram-negative); and Candida albicans (ATCC 36082, fungus). In brief,organisms were cultured to logarithmic phase and inoculated at a densityof 10⁶ colony forming units/ml in buffered molecular grade agarose atthe indicated pH. Five μg of peptide resuspended in sterile deionizedwater were introduced into wells formed in the underlay, and incubatedfor 3 h at 37° C. Nutrient-containing overlay medium was then applied,and assays incubated at 37° C. or 30° C. for bacteria or fungi,respectively. Defensin HNP-1 was tested in parallel as a standardcontrol. After 24 h, zones of complete or partial inhibition weremeasured. All assays were repeated independently a minimum of two times.Tang et al., Infect. Immun 70:6524 (2002) for detailed methodology.].

As predicted by the signature model, brazzein and charybdotoxin exerteddirect antimicrobial activity against bacteria and C. albicans (FIG. 7).Notably, these peptides exhibited pH-specific antimicrobial activities,which in some conditions exceeded that of HNP-1. These resultsdemonstrate for the first time to our knowledge the direct antimicrobialactivities of brazzein and charybdotoxin. In contrast, metallothioneinII failed to exert antimicrobial activity against any organism testedunder any condition, as predicted by the model.

An alternative approach was also used to validate the multidimensionalsignature model. Tachyplesins are known cysteine-containingantimicrobial peptides from the horseshoe crab, Tachypleus. Twotachyplesins were retrieved from protein database searches employing thelevomeric sequence formula (Table III). The model predicted that,because they have known antimicrobial activity, and fulfill the primarysequence criteria, tachyplesins would contain a γ-core motif. The3-dimensional structure of tachyplesin I became available subsequent todevelopment of the model (Laederach et al., Biochem. 41:12359 (2002)),and as predicted, exhibits a γ-core motif integral to themultidimensional signature of disulfide-containing antimicrobialpeptides (FIG. 6). Confirmation of the 3-dimensional γ-core structurefrom antimicrobial activity and primary sequence pattern offers a robustand complementary validation of the multidimensional signature model.

The phylogenetic relationships among antimicrobial peptides containingthe multidimensional signature were also examined Study peptides sortedin a continuum of increasing structural complexity relative to theγ-core motif, rather than evolutionary relatedness of the sourceorganisms (FIG. 8 A). This phylogenetic pattern is consistent withconservation of the γ-core motif amongst cysteine-containingantimicrobial peptides across biological kingdoms.

Example III Validation of the Multidimensional Antimicrobial PeptideSignature Model

This example demonstrates the discovery of iterations of the γ-coremotif in kinocidins and validation of the antimicrobial signature modelin human IL-8.

A bioinformatics approach was used to specify and compare phylogeny andhomology among kinocidin iterations of the γ-core motif previouslyidentified in proteins with known or predicted antimicrobial function(Yount, N. Y. & Yeaman, M. R. (2004) Proc Natl Acad Sci USA 101,7363-8). The kinocidin γ-core (γ_(KC) core) signature is an iteration ofthe antimicrobial peptide γ-core (γ_(AP)), conforming to ananti-parallel β-hairpin comprised of a 13-17 amino acid pattern with acentral hydrophobic region typically flanked by basic residues. Theγ_(KC) core motif can be characterized by the following consensussequence formula:

(SEQ ID NO: 363) NH2...[X8-11]-[GX3C]-[X2]-[P]...COOH

Sequence and 3-D analyses of more than 30 human CXC and CC kinocidinsdemonstrated that the γ_(KC) core corresponds to the most highlyconserved domain within the mature portion of these proteins (FIG. 12).Notably, the cysteine array and glycine residues hallmark of the γ_(AP)core are also conserved in the γ_(KC) motif. In kinocidins, the GXCconsensus of the γ_(AP) core is adapted such that a GX₃C pattern isoften observed. While the initial glycine of the GX3C pattern ofkinocidins is conserved (>60%), the requirement for a glycine in thisposition is not absolute, with uncharged hydrophilic residues (A or N)as most common substitutions. A proline residue at its C-terminal aspectis another highly conserved feature (>95%) of the γ_(KC) motif. Thisresidue is located immediately prior to, and likely initiates theensuing α-helical domain (FIG. 12).

Human IL-8 contains the sequence NH2 . . . CANTEIIVKLSDGRELCLDP . . .COOH (SEQ ID NO: 40), representing the γ_(KC) core consensus formula,and shares specific physicochemical patterns of amphipathicity, chargedistribution, and proline positioning with known kinocidins (FIG. 12).Based on the extensive structural homologies to kinocidins, IL-8 waspredicted to have direct antimicrobial efficacy.

To confirm that IL-8 exerts direct antimicrobial efficacy, antimicrobialassays using a panel of prototypic pathogens were conducted as describedin the following paragraphs.

Briefly, the antimicrobial assays were performed against a panel ofprototype human pathogens as previously detailed (Yoshimura, T.,Matsushima, K., Tanaka, S., Robinson, E. A., Appella, E., Oppenheim, J.J. & Leonard, E. J. (1987) Proc Natl Acad Sci USA 84, 9233-7):Staphylococcus aureus (ATCC 27217; Gram-positive bacterium); Salmonellatyphimurium (5996s; Gram-negative bacterium); and the fungus Candidaalbicans (ATCC 36082). Defensin HNP-1 was included in each assay as aninternal control, and all assays were conducted a minimum of twoindependent times. Results of independent assays were analyzed usingWilcoxon Rank Sum analysis with Bonferroni correction for multiplecomparisons.

For the Solid-Phase Assay, mid-logarithmic phase organisms wereprepared, introduced into buffered agarose (PIPES [10 mM, pH 7.5] or MES[2.0 mM, pH 5.5]) to achieve final inocula of 10⁶ CFU/ml, and pouredinto plates. Peptides (0.5 nmol/well [50 nmol/ml]) were added to wellsin the seeded matrix, and incubated for 3 h at 37° C. Nutrient overlaymedium was applied, and assays incubated at 37° C. or 30° C. forbacteria or fungi, respectively. After 24 h, zones of inhibition weremeasured, and results recorded as zones of complete or partialinhibition. This assay reflects microbiostatic (inhibition) and/ormicrobiocidal (killing) activities, but does not distinguish theseeffects.

As predicted by biophysical and structural congruence with otherkinocidins, IL-8 exerted antimicrobial activity against bacteria andfungi in the solid-phase assay at pH 5.5 and 7.5. In many cases,antimicrobial spectra and efficacy of IL-8 were greater than other studykinocidins (FIG. 14). IL-8 was equivalent to lymphotactin, but moreefficacious than any other test kinocidin against S. typhimurium.Against S. aureus, IL-8 (pH 5.5) had modest activity, with RANTES (pH7.5), and lymphotactin (pH 7.5) being more efficacious. Significantly,IL-8 possessed striking antifungal activity versus C. albicans, greaterthan any other kinocidin at pH 5.5, and the only kinocidin withanti-candidal activity at pH 7.5. Excepting S. aureus, IL-8 displayedcomparable antimicrobial efficacy to the classical antimicrobial peptideHNP-1 under the conditions tested. In addition to IL-8, the theseresults also demonstrate a direct antimicrobial efficacy for thekinocidin MCP-1. Interestingly, while MCP-1 exerted significant activityagainst Gram-positive and -negative bacteria, it had no measurableantifungal activity (FIG. 14).

Recombinant human chemokines [IL-8, RANTES, GRO-α, MCP-1, PF-4, andlymphotactin (Biosource International, Camarillo, Calif.)] and humanneutrophil defensin-1 [HNP-1 (Peptides International, Louisville, Ky.)]were obtained commercially. Structural domains of IL-8 were generated byF-moc solid-phase synthesis: γ-core (ANTEIIVKLSDGRELCLDP; IL-8γ (SEQ IDNO: 42)), and α-helix (KENWVQRVVEKFLKRAENS; IL-8α (SEQ ID NO: 1)).Peptides were purified by RP-HPLC as previously described ((Tang, Y. Q.,Yeaman, M. R. & Selsted, M. E. (2002) Infect Immun 70, 6524-33); >95%purity), and authenticated by amino acid analysis (Molecular StructureFacility, University of California, Davis) and MALDI-TOF spectrometry(UCLA Spectrometry Facility). Experimentally determined masses werewithin standard confidence intervals (<±0.1% of calculated molecularweight).

To characterize IL-8's antimicrobial activity in more detail, aquantitative solution-phase assay was carried out at pH 5.5 and 7.5.Organisms were prepared and adjusted to 10⁶ CFU/ml in PIPES or MESbuffer as above but lacking agarose, and dispensed (5×10⁴ CFU per 50 μlaliquot). Peptide (concentration range, 20-0.00125 nmol/ml) wasintroduced into the assay medium, and incubated for 1 hour at 37° C.After incubation, media were serially diluted and plated in triplicatefor enumeration. IL-8 was highly fungicidal for C. albicans, achieving afive-log reduction in surviving CFU with 2.5 nmol/ml (20 μg/ml) peptideat pH 5.5 within 1 hour (FIG. 15). IL-8 was also fungicidal for C.albicans at pH 7.5, with a 2-log reduction in surviving CFU over 1 hourat a concentration of 2.5 nmol/ml. Interestingly, IL-8 did not exertmicrobicidal activity against S. typhimurium or S. aureus in this assay,indicating that its efficacies versus these organisms in solid-phaseassay were due to bacteriostatic effects.

Synthetic IL-8γ and IL-8α peptides were used to probe for moleculardeterminants of IL-8 antimicrobial activity. IL-8α displayed a spectrumof efficacy virtually indistinguishable from that of the native molecule(FIG. 14). IL-8γ had no detectable antimicrobial efficacy at either pH5.5 or 7.5. When assayed in combination, the pattern of antimicrobialactivity was identical to that of IL-8α alone, indicating that IL-8γ didnot impede the antimicrobial efficacy of IL-8α.

In the solution-phase assay, as little as 0.125 nmol/ml (1.0 μg/ml) ofIL-8α achieved a five-log reduction in surviving C. albicans at pH 5.5in 1 hour exposure (FIG. 15). By mass, the autonomous IL-8α domainconferred a 10-fold greater activity than native IL-8 against thisorganism. The pH specific efficacy patterns of IL-8α also mirrored thoseof full-length (FIG. 15). Consistent with results of the solid-phaseassay, IL-8γ had no measurable activity in the solution-phase assay.

To assess the physiological relevance of the observed in vitro (solidand solution-phase) antimicrobial activity for IL-8 and IL-8α, weassessed the efficacy of IL-8α in the ex vivo biomatrix assay. Thisassay has been developed to assess antimicrobial polypeptide efficacy incomplex human blood matrices. Antimicrobial activity of IL-8α in humanwhole blood and homologous plasma and serum fractions was assessed(Yeaman, M. R., Gank, K. D., Bayer, A. S., and Brass, E. P. (2002)Antimicrob Agents Chemother 46, 3883-3891). For biomatrix studies, thewell-characterized Escherichia coli strain ML-35 was used as the targetorganism (Lehrer, R. I., Barton, A., Daher, K. A., Harwig, S. S., Ganz,T., and Selsted, M. E. (1989) J Clin Invest 84, 553-561). This strain isresistant to serum, ideal for use in assessing peptide antimicrobialactivity in blood and blood-derived matrices (Yeaman, M. R., Gank, K.D., Bayer, A. S., and Brass, E. P. (2002) Antimicrob Agents Chemother46, 3883-3891). Organisms were cultured to mid-logarithmic phase inbrain-heart infusion broth (Difco Laboratories, Detroit, Mich.) at 37°C., washed, and resuspended in PBS (Irvine Scientific; pH 7.2). Inoculawere quantified spectrophotometrically and validated by quantitativeculture. Biomatrices were distributed in 85-μl aliquots into 96-wellmicrotiter plates (Corning Glass Works, Corning, N.Y.) Peptide (5 μl;concentration range 1.0-50.0 μg/ml) was added either simultaneously withthe microorganism (10 μl; 10⁵ CFU/ml), or after a 120 min pre-incubationperiod in the biomatrix. The mixtures were incubated with constantagitation for 2 h at 37° C. After incubation, aliquots were diluted andquantitatively cultured in triplicate onto blood agar. Survivingorganisms were enumerated as CFU/ml. Experiments were performed aminimum of two independent times on different days and with differentblood donor sources.

Importantly, IL-8α demonstrated significant efficacy against E. coli,causing decreases of up to log 5 CFU/ml at 10 μg/ml peptide. Greatestefficacy was seen in whole blood and serum in co-incubation studies,with less activity in plasma fractions or after 2 hour preincubation.

To gain insights into these antimicrobial profiles, the structures ofIL-8γ and IL-8α were investigated using biophysical and computationalmethods.

Secondary structures of IL-8 peptide domains were assessed by circulardichroism (CD) as previously noted (Sheppard et al. (2004) J Biol Chem279, 30480-30489). Spectra were recorded with an AVIV 62DSspectropolarimeter (Aviv Biomedical Inc.). In brief, the purifiedpeptides were solubilized (50 μg/ml in 50 mM NH4HC03; pH 5.5 or 7.5) andscanned using a 0.1-mm light path from 260 to 185 nm (rate, 10 nm/min;sample interval, 0.2 nm; 25° C.). A mean of 8 buffer-subtracted spectrawere deconvoluted into helix, β-sheet, turn, and extended structuresusing Selcon (Sreerama et al. (1999) Protein Sci 8, 370-380) andDichroweb (Lobley et al., (2002) Bioinformatics 18, 211-212);cryst.bbk.ac.uk/cdweb) as indicated (Sheppard et al. (2004) J Biol Chem279, 30480-30489; Surewicz and Mantsch (1988) Biochim Biophys Acta 952,115-130; Goormaghtigh et al. (1999) Biochim Biophys Acta 1422, 105-185).

Protein structure data were obtained from the National Center forBiotechnology Information (NCBI; PubMed Database), and visualized usingProtein Explorer (Martz, E. (2002) Trends Biochem Sci 27, 107-109).Structural alignments were carried out using combinatorial extension,and significance of homology assessed by root mean square deviationanalysis, as previously described (Yount and Yeaman (2004) Proc NatlAcad Sci USA 101, 7363-7368; Shindyalov and Bourne (1998) Protein Eng11, 739-747). 3CD spectrometry indicated that IL-8γ and IL-8αrecapitulated structures of corresponding regions in full-length IL-8(FIG. 16). IL-8γ exhibited spectra consistent with a β-sheet structure,suggesting it spontaneously adopts a fold similar to that in nativeIL-8. Likewise, IL-8α displayed classic double dichroic minima at 208and 218 nm, hallmark of α-helices, and concordant with the correspondingregion in IL-8. These data suggest the forces responsible for secondarystructures of these domains function independently fromcysteine-stabilization or other constraints acting within the nativemolecule. Moreover, each structure was stable at pH 5.5 and pH 7.2 (FIG.16).

To complement spectrometric studies, 3-D models of IL-8γ and IL-8α werecreated using homology and energy-based methods. Three-dimensionalmodels of IL-8 domains were created using complementary methods (Yountet al. (2004) Antimicrob Agents Chemother 48, 4395-404). Homology(SWISSMODEL, BLAST2P; (Godzik et al. (1992) J Mol Biol 227, 227-38;Jaroszewski et al. (1998) Protein Sci 7, 1431-40], dynamic alignment(SIM) (Huang, X., and Miller, W. (1991) Adv. Appl. Math. 12, 337-367)and refined match (ProModIII) algorithms were used to identify modelingtemplates. In a parallel strategy, IL-8 amino acid sequences wereconverted to putative solution conformations by threading methods(Matchmaker [Godzik et al. (1992) J Mol Biol 227, 227-238]; Gene-Fold[Jaroszewski et al. (1998) Protein Sci 7, 1431-1440]) implemented withSYBYL software (Tripos Associates, St. Louis, Mo.). Target conformerswere refined using AMBER95 force field and molecular dynamics (Cornellet al. (1995) J Am Chem Soc 117, 5179-5197). In alternative approaches,molecular dynamics were executed without 0.4 kJ constraint penalties forcanonical Ramachandran φ and ψ angles.

The template utilized for target peptide modeling was human IL-8 (PDBcode, 1IL8). As expected, each peptide retained secondary structurecorresponding to homologous domains within the native molecule (FIG.17). The IL-8γ core motif displayed an anti-parallel β-sheet motif,while the preferential conformation for IL-8α was a highly stableα-helical motif comprised of four turns. These structure assignments arestrongly supported by favorable empirical energy functions, equivalentto those of the IL-8 template.

TABLE III Comparative physicochemical properties of human kinocidins andα-helical domains thereof. Classification Schema Native Moleculeα-Helical Domain Class Ligand ID Name AA M Q pI AA_(α) Q_(α) M_(α)pI_(α) H_(α) CXC CXCL8 IL-8 71 8299 +4 9.0 17 +2 2103 10.0 6.70 CXCCXCL4 PF-4 70 7769 +3 8.8 13 +3 1573 9.8 6.12 CXC CXCL1 GRO-α 72 7751 +69.5 16 +2 1843 9.6 4.71 CC CCL2 MCP-1 76 8685 +6 9.4 19 0 2287 6.8 5.35CC CCL5 RANTES 68 7851 +5 9.2 13 0 1655 6.1 6.71 C CL1 Lymphotactin 9210173 +9 10.6 14 +2 1735 10.7 3.73 Physicochemical parameters areabbreviated for the native or α-helical domain (α),: AA—amino acids;M—average mass (Da); Q—calculated charge at pH 7.0; pI—estimatedisoelectric point (Bjellqvist et al., (1994) Electrophoresis 15,529-39); H—hydrophobic moment (Zidovetzki et al. (2003) Biophys Chem100, 555-75).

Throughout this application various publications have been referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific examples and studies detailed above are onlyillustrative of the invention. It should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A kinocidin peptide comprising a C-terminal portion amino acidsequence of a kinocidin, wherein said C-terminal portion comprises anα-helical secondary structure, wherein said C-terminal portion furthercomprises antimicrobial activity, and wherein said kinocidin comprises aγ_(KC) core.
 2. The kinocidin peptide of claim 1, wherein the kinocidinis a CXC, CX₃C, CC, or C class chemokine.
 3. The kinocidin peptide ofclaim 1, wherein said amino acid sequence is KENWVQRVVEKFLKRAENS (SEQ IDNO: 1).
 4. The kinocidin peptide of claim 1, wherein said amino acidsequence is QAPLYKKIIKKLLES (SEQ ID NO: 2).
 5. The kinocidin peptide ofclaim 1, wherein said amino acid sequence is ASPIVKKIIEKMLNSDKSN (SEQ IDNO: 3).
 6. The kinocidin peptide of claim 1, wherein said amino acidsequence is selected from the group depicted in FIG.
 21. 7. Thekinocidin peptide of claim 1, wherein said alpha-helical secondarystructure comprises between 10 and 35 amino acids.
 8. The kinocidinpeptide of claim 1, wherein said alpha-helical secondary structurecomprises a mass between 1100 Da and 3850 Da.
 9. The kinocidin peptideof claim 1, wherein said alpha-helical secondary structure comprises acalculated charge between 0 and (+) 5 at pH 7.0.
 10. The kinocidinpeptide of 1, wherein said alpha-helical secondary structure comprisesan estimated isoelectric point between 5 and
 15. 11. The kinocidinpeptide of claim 1, wherein said alpha-helical secondary structurecomprises a hydrophobic moment between 3 and
 8. 12. A method fortreating an infectious disease or condition in a subject in need of suchtreatment comprising administering to the subject an effective amount ofa kinocidin peptide of claim 1.